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Microfluidic and Nanofluidic Intracellular Delivery.
Innate cell function can be artificially engineered and reprogrammed by introducing biomolecules, such as DNAs, RNAs, plasmid DNAs, proteins, or nanomaterials, into the cytosol or nucleus. This process of delivering exogenous cargos into living cells is referred to as intracellular delivery. For instance, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing begins with internalizing Cas9 protein and guide RNA into cells, and chimeric antigen receptor-T (CAR-T) cells are prepared by delivering CAR genes into T lymphocytes for cancer immunotherapies. To deliver external biomolecules into cells, tools, including viral vectors, and electroporation have been traditionally used; however, they are suboptimal for achieving high levels of intracellular delivery while preserving cell viability, phenotype, and function. Notably, as emerging solutions, microfluidic and nanofluidic approaches have shown remarkable potential for addressing this open challenge. This review provides an overview of recent advances in microfluidic and nanofluidic intracellular delivery strategies and discusses new opportunities and challenges for clinical applications. Furthermore, key considerations for future efforts to develop microfluidics- and nanofluidics-enabled next-generation intracellular delivery platforms are outlined.
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CRISPR/Cas9-mediated genome editing of the fatty acid desaturase 2 gene in Brassica napus.
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated genome editing system has been widely applied as a powerful tool for modifying preferable endogenous genes. This system is highly expected to be further applied for the breeding of various agronomically important plant species. Here we report the modification of a fatty acid desaturase 2 gene (FAD2), which encodes an enzyme that catalyzes the desaturation of oleic acid, in Brassica napus cv. Westar using the CRISPR/Cas9 system. Two guide RNAs were designed for BnaA.FAD2.a (FAD2_Aa). Of 22 regenerated shoots with FAD2_Aa editing vectors, three contained mutant alleles. Further analysis revealed that two of three mature plants (Aa1#13 and Aa2#2) contained the mutant alleles. The mutant fad2_Aa allele had a 4-bp deletion, which was inherited by backcross progenies (BC1) in the Aa1#13 line. Furthermore, plants with the fad2_Aa allele without transgenes were selected from the BC1 progenies and plants homozygous for fad2_Aa were then produced by self-crossing these BC1 progenies (BC1S1). Fatty acid composition analysis of their seeds revealed a statistically significant increase in the content of oleic acid compared with that in wild-type seeds. These results showed that the application of the CRISPR/Cas9 system is useful to produce desirable mutant plants with an agronomically suitable phenotype by modifying the metabolic pathway in B. napus.
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A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria.
Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection.
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Highly efficient CRISPR/Cas9-RNP mediated CaPAD1 editing in protoplasts of three pepper (Capsicum annuum L.) cultivars.
Parthenocarpy, characterized by seedless fruit development without pollination or fertilization, offers the advantage of consistent fruit formation, even under challenging conditions such as high temperatures. It can be induced by regulating auxin homeostasis; PAD1 (PARENTAL ADVICE-1) is an inducer of parthenocarpy in Solanaceae plants. However, precise editing of PAD1 is not well studied in peppers. Here, we report a highly efficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) for CaPAD1 editing in three valuable cultivars of pepper (Capsicum annuum L.): Dempsey, a gene-editable bell pepper; C15, a transformable commercial inbred line; and Younggo 4, a Korean landrace. To achieve the seedless pepper trait under high temperatures caused by unstable climate change, we designed five single guide RNAs (sgRNAs) targeting the CaPAD1 gene. We evaluated the in vitro on-target activity of the RNP complexes in three cultivars. Subsequently, we introduced five CRISPR/Cas9-RNP complexes into protoplasts isolated from three pepper leaves and compared indel frequencies and patterns through targeted deep sequencing analyses. We selected two sgRNAs, sgRNA2 and sgRNA5, which had high in vivo target efficiencies for the CaPAD1 gene across the three cultivars and were validated as potential off-targets in their genomes. These findings are expected to be valuable tools for developing new seedless pepper cultivars through precise molecular breeding of recalcitrant crops in response to climate change.
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CRISPR/Cas9-Mediated Generation of Tetracycline Repressor-Based Inducible Knockdown in Toxoplasma gondii.
The phylum Apicomplexa groups numerous pathogenic protozoan parasites including Plasmodium, the causative agent of malaria, Cryptosporidium which can cause severe gastrointestinal infections, as well as Babesia, Eimeria, and Theileria that account for considerable economic burdens to poultry and cattle industry. Toxoplasma gondii is the most ubiquitous and opportunistic member of this phylum able to infect all warm-blooded animals and responsible for severe disease in immunocompromised individuals and unborn fetuses.Due to its ease of cultivation and genetic tractability T. gondii has served as recipient for the transfer and adaptation of multiple genetic tools developed to control gene expression. In these parasites, a collection of tight conditional systems exists to control gene expression at the levels of transcription, RNA degradation or protein stability. The recent implementation of the CRISPR/Cas9 technology considerably reduces time and effort to generate transgenic parasites and at the same time increases to an ultimate level of precision the editing of the parasite genome. Here, we provide a step-by-step protocol for CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knockdown in T. gondii.
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Recent advances in the use of the CRISPR-Cas system for the detection of infectious pathogens.
Infectious diseases cause great economic loss and individual and even social anguish. Existing detection methods lack sensitivity and specificity, have a poor turnaround time, and are dependent on expensive equipment. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has been widely used in the detection of pathogens that cause infectious diseases owing to its high specificity, sensitivity, and speed, and good accessibility. In this review, we discuss the discovery and development of the CRISPR-Cas system, summarize related analysis and interpretation methods, and discuss the existing applications of CRISPR-based detection of infectious pathogens using Cas proteins. We conclude the challenges and prospects of the CRISPR-Cas system in the detection of pathogens.
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Perspectives on the use of the CRISPR system in plants to improve recombinant therapeutic protein production.
The plant-based system is a promising platform for producing biotherapeutics due to its scalability, cost-effectiveness, and lower risk of contamination by human pathogens. However, several challenges remain, including optimizing yield, stability, functionality, and the immunogenic properties of recombinant proteins. In this context, this review explores the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology to improve the production of recombinant therapeutic proteins in plants. Traditional tools and strategies for plant-based recombinant protein production are discussed, highlighting their limitations and the potential of CRISPR to overcome these boundaries. It delves into the components of the CRISPR-Cas system and its application in optimizing therapeutic protein function and yield. Major strategies include modifying glycosylation patterns to humanize plant-produced proteins, metabolic pathway engineering to increase protein accumulation, and the precise integration of transgenes into specific genomic loci to enhance expression stability and productivity. These advancements demonstrate how CRISPR system can overcome bottlenecks in plant molecular farming and enable the production of high-quality therapeutic proteins. Lastly, future trends and perspectives are examined, emphasizing ongoing innovations and challenges in the field. The review underscores the potential of CRISPR to reshape plant biotechnology and support the growing demand for recombinant therapeutics, offering new avenues for sustainable and efficient protein production systems. KEY MESSAGE: CRISPR technology has the potential to improve plant-based therapeutic protein production by optimizing yield, stability, and humanization, overcoming bottlenecks, and enabling sustainable, efficient systems for recombinant biotherapeutics.
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Nanoparticle-mediated delivery of non-viral gene editing technology to the brain.
Neurological disorders pose a significant burden on individuals and society, affecting millions worldwide. These disorders, including but not limited to Alzheimer's disease, Parkinson's disease, and Huntington's disease, often have limited treatment options and can lead to progressive degeneration and disability. Gene editing technologies, including Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats-associated Protein 9 (CRISPR-Cas9), offer a promising avenue for potential cures by targeting and correcting the underlying genetic mutations responsible for neurologic disorders. However, efficient delivery methods are crucial for the successful application of gene editing technologies in the context of neurological disorders. The central nervous system presents unique challenges to treatment development due to the blood-brain barrier, which restricts the entry of large molecules. While viral vectors are traditionally used for gene delivery, nonviral delivery methods, such as nanoparticle-mediated delivery, offer safer alternatives that can efficiently transport gene editing components. Herein we aim to introduce the three main gene editing nucleases as nonviral treatments for neurologic disorders, the delivery barriers associated with brain targeting, and the current nonviral techniques used for brain-specific delivery. We highlight the challenges and opportunities for future research in this exciting and growing field that could lead to blood-brain barrier bypassing therapeutic gene editing.
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Probing the impact of chromatin conformation on genome editing tools.
Transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases derived from clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems have become ubiquitous genome editing tools. Despite this, the impact that distinct high-order chromatin conformations have on these sequence-specific designer nucleases is, presently, ill-defined. The same applies to the relative performance of TALENs and CRISPR/Cas9 nucleases at isogenic target sequences subjected to different epigenetic modifications. Here, to address these gaps in our knowledge, we have implemented quantitative cellular systems based on genetic reporters in which the euchromatic and heterochromatic statuses of designer nuclease target sites are stringently controlled by small-molecule drug availability. By using these systems, we demonstrate that TALENs and CRISPR/Cas9 nucleases are both significantly affected by the high-order epigenetic context of their target sequences. In addition, this outcome could also be ascertained for S. pyogenes CRISPR/Cas9 complexes harbouring Cas9 variants whose DNA cleaving specificities are superior to that of the wild-type Cas9 protein. Thus, the herein investigated cellular models will serve as valuable functional readouts for screening and assessing the role of chromatin on designer nucleases based on different platforms or with different architectures or compositions.
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Whole-Genome Sequencing Reveals Rare Off-Target Mutations in MC1R-Edited Pigs Generated by Using CRISPR-Cas9 and Somatic Cell Nuclear Transfer.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used to create animal models for biomedical and agricultural use owing to its low cost and easy handling. However, the occurrence of erroneous cleavage (off-targeting) may raise certain concerns for the practical application of the CRISPR-Cas9 system. In this study, we created a melanocortin 1 receptor (MC1R)-edited pig model through somatic cell nuclear transfer (SCNT) by using porcine kidney cells modified by the CRISPR-Cas9 system. We then carried out whole-genome sequencing of two MC1R-edited pigs and two cloned wild-type siblings, together with the donor cells, to assess the genome-wide presence of single-nucleotide variants and small insertions and deletions (indels) and found only one candidate off-target indel in both MC1R-edited pigs. In summary, our study indicates that the minimal off-targeting effect induced by CRISPR-Cas9 may not be a major concern in gene-edited pigs created by SCNT.
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