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Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds.
CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities.
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CRISPR-Cas13 technology portfolio and alliance with other genetic tools.
The exploitation of CRISPR-Cas systems especially CRISPR-Cas9 has led to radical advances in genome editing, gene activation, gene repression, protein imaging, and beyond. However, these applications are limited to targeting of DNA rather than RNA. CRISPR-Cas13 is the first reported CRISPR system targeting RNA and thus opens a new avenue for transcription regulation. While a plethora of reviews have systematically documented CRISPR-Cas9 toolbox, this review focuses on CRISPR-Cas13 family, covering aspects of classification, structures, response to foreign invaders, and genetic toolbox. In particular, we compare CRISPR-Cas13 with other RNA regulation tools such as RNA interference and antisense RNA technology to ponder the possibility of combining them to engineer hierarchical regulatory networks fulfilling novel functions. Lastly, we summarize the wide applications of CRISPR-Cas13 toolbox and the status quo that requires amelioration. Overall, this review charts a landscape of CRISPR-Cas13 technology portfolio to provide insights for gene regulation, metabolic engineering and synthetic biology.
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CRISPR-Mediated Construction of Gene-Knockout Mice for Investigating Antiviral Innate Immunity.
With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity.
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Rapid assessment of CRISPR gRNAs with optimized protoplast transformation in Maize.
We developed an optimized CRISPR/Cas9 gene editing system using maize mesophyll protoplasts to enable rapid evaluation of guide RNA (gRNA) activity. Using the tropical inbred line Tzi8, we improved protoplast isolation and transfection protocols, achieving high yields of 17.88 × 10⁶ viable protoplasts per gram fresh weight while extending post-transfection viability. Etiolated seedlings and vertical leaf cutting significantly enhanced protoplast recovery and viability. A transfection efficiency of ~ 50% was achieved using 10 µg of plasmid DNA; higher DNA inputs did not result in significant gains, resulting in a more resource-efficient approach. Protoplast viability was maintained for up to seven days post-transfection, allowing for downstream applications that require extended incubation. This optimized system was used to assess the editing efficiency of nine gRNAs targeting three key floral repressors (ZmCCT9, ZmCCT10, and ZmRap2.7) across four maize genotypes (Tzi8, CML277, B73, and B104). These floral repressor genes are involved in the photoperiod sensitivity of tropical maize, a major challenge in the effort to introduce tropical maize germplasm into temperate breeding programs. Editing efficiencies ranged from 0.4% to 23.7%, with some variation observed between gRNAs and genotypes. Although protoplast-based assays do not currently enable plant regeneration, this platform offers a rapid method for in vivo gRNA validation, reducing assay time from months to days. This work expands the gene editing toolkit for tropical maize, supporting efforts to overcome breeding barriers through gene editing.
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Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.
Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells. As an example, we describe how to knock out the glutamine synthetase (GS) gene, which increases the selection efficiency of the GS-mediated gene amplification system.
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Temporal and rheostatic control of genome editing with a chemically-inducible Cas9.
Nuclease-mediated DNA cleavage and subsequent repair lie at the heart of genome editing, and the RNA-guided endonuclease Cas9 has emerged as the most widely-used tool for facilitating this process. Extensive biochemical and biophysical efforts have revealed much regarding the structure, mechanism, and cellular properties of Cas9. This has enabled engineering of Cas9 variants with enhanced activity, specificity, and other features. However, we lack a detailed understanding of the kinetics of Cas9-mediated DNA cleavage and repair in vivo. To study in vivo Cas9 cleavage kinetics and activity dose-dependence, we have engineered a chemically-inducible, single-component Cas9, ciCas9. ciCas9 allows for temporal and rheostatic control of Cas9 activity using a small molecule activator, A115. We have also developed a droplet-digital PCR-based assay (DSB-ddPCR) to directly quantify Cas9-mediated double-stranded breaks (DSBs). The methods in this chapter describe the application of ciCas9 and DSB-ddPCR to study the kinetics and dose-dependence of Cas9 editing in vivo.
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Bioorthogonal Reaction-Mediated Tumor-Selective Delivery of CRISPR/Cas9 System for Dual-Targeted Cancer Immunotherapy.
CRISPR system-assisted immunotherapy is an attractive option in cancer therapy. However, its efficacy is still less than expected due to the limitations in delivering the CRISPR system to target cancer cells. Here, we report a new CRISPR/Cas9 tumor-targeting delivery strategy based on bioorthogonal reactions for dual-targeted cancer immunotherapy. First, selective in vivo metabolic labeling of cancer and activation of the cGAS-STING pathway was achieved simultaneously through tumor microenvironment (TME)-biodegradable hollow manganese dioxide (H-MnO2 ) nano-platform. Subsequently, CRISPR/Cas9 system-loaded liposome was accumulated within the modified tumor tissue through in vivo click chemistry, resulting in the loss of protein tyrosine phosphatase N2 (PTPN2) and further sensitizing tumors to immunotherapy. Overall, our strategy provides a modular platform for precise gene editing in vivo and exhibits potent antitumor response by boosting innate and adaptive antitumor immunity.
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Gene Editing: A New Tool for Viral Disease.
The emergence of the CRISPR/Cas system of antiviral adaptive immunity in bacteria as a facile system for gene editing in mammalian cells may well lead to gene editing becoming a novel treatment for a range of human diseases, especially those caused by deleterious germline mutations. Another potential target for gene editing are DNA viruses that cause chronic pathogenic diseases that cannot be cured by using currently available drugs. We review the current state of this field and discuss the potential advantages and problems with using a gene editing approach as a treatment for diseases caused by DNA viruses.
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Protocol Development for CRISPR/Cas9 Knockout of the Anti-inflammatory Protein TNIP1 in HaCaT Keratinocytes.
Gene editing in cultured cells, including the intent of sequence disruption to achieve a functional knockout of the targeted gene, has been greatly facilitated with the advent of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) technology. Primary cell strains and immortalized cell lines from diverse tissue types have been successfully targeted both for basic research examining the effects of loss of the correlating protein and for modeling select loss-of-function disorders. Such accomplishments have extended to cutaneous cells, especially epidermal keratinocytes given their key structural and functional role in barrier formation and surveillance of and response to surface events such as triggering and processing inflammatory responses. Here we describe disruption of the Tumor Necrosis factor-induced protein 3-Interacting Protein 1 (TNIP1) gene in human HaCaT keratinocytes to generate an ongoing loss of expression as a parallel system to transient knockdown we had previously achieved with siRNA transfection. The TNIP1 protein restricts cytoplasmic progression of inflammatory signals. We cover our CRISPR/Cas9 vector choice, enrichment of transfected cells via positive selection for puromycin resistance, their subsequent cloning, and gene disruption and expression analysis. We also emphasize prior keratinocyte-CRISPR/Cas9 literature as a springboard for other investigators and to illustrate the widespread relevance of such editing to the diverse, yet highly consequentially different, genes expressed in keratinocytes.
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Therapeutic Targeting in Ovarian Cancer: Nano-Enhanced CRISPR/Cas9 Gene Editing and Drug Combination Therapy.
Ovarian cancer is the third most common gynecological cancer worldwide. Due to the high recurrence rate of advanced-stage ovarian cancer, often resulting from drug-resistant and refractory disease, various treatment strategies are under investigation. Genome editing of therapeutic target genes holds promise in enhancing cancer treatment efficacy by elucidating gene functions and mechanisms involved in cancer progression. The CRISPR/Cas9 system, in particular, shows great potential in ovarian cancer gene therapy and drug development. Targeting therapeutic genes such as BRCA1/2, P53, Snai1 etc, could improve the therapeutic strategy in ovarian cancer. CRISPR/Cas9 is a powerful gene-editing tool that there are many on-going clinical trials to treat various diseases including cancer. Nano-based delivery systems for CRISPR/Cas9 offer further therapeutic benefits, leveraging the unique properties of nanoparticles to improve delivery efficiency. Nano-based delivery systems could enhance the stability of CRISPR/Cas9 delivery formats (such as plasmid, mRNA, etc) and improve the delivery precision of delivery to target tumors. Additionally, combining CRISPR/Cas9 with targeted drug treatments, especially those aimed at genes associated with drug resistance, may significantly improve therapeutic outcomes in ovarian cancer. In this review, we discuss therapeutic target genes and their mechanisms in ovarian cancer, advances in nano-based CRISPR/Cas9 delivery, and the therapeutic potential of combining CRISPR/Cas9 with drug treatments for ovarian cancer.
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