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Gene Editing in the Chagas Disease Vector Rhodnius prolixus by Cas9-Mediated ReMOT Control.
Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. However, transgenesis and gene editing protocols to advance the field are still lacking. Here, we tested protocols for the maternal delivery of CRISPR-Cas9 (clustered regularly spaced palindromic repeats/Cas-9 associated) elements to developing R. prolixus oocytes and strategies for the identification of insertions and deletions (indels) in target loci of resulting gene-edited generation zero (G0) nymphs. We demonstrate successful gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) with the ovary-targeting BtKV ligand. These results provide proof of concepts for generating somatic mutations in R. prolixus and potentially for generating germ line-edited lines in triatomines, laying the foundation for gene editing protocols that could lead to the development of novel control strategies for vectors of Chagas disease.
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The expanding toolbox for genome engineering.
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Generating adamts13-/- Zebrafish via CRISPR/Cas9 Gene Editing.
The zebrafish (Danio rerio) is a powerful vertebrate model for studying hematologic and thrombotic diseases due to its genetic tractability and conservation of hemostatic pathways with humans. In this chapter, we describe a detailed workflow for generating adamts13 knockout zebrafish using CRISPR/Cas9-mediated genome editing. Methods include the design and preparation of guide RNAs, Cas9 mRNA synthesis, and embryo microinjection at the one-cell stage. Alternative strategies for gRNA generation, including synthetic crRNA:tracrRNA duplexes, are also outlined. We provide protocols for screening founder fish, genotyping, and establishing stable mutant lines through outcrossing, thereby minimizing potential off-target effects. Practical notes on embryo handling, RNA stability, and contamination prevention are highlighted to ensure reproducibility. Together, these procedures establish a robust framework for creating zebrafish loss-of-function models, enabling mechanistic studies of ADAMTS13 function in vivo and advancing the exploration of thrombotic disease pathophysiology. This approach can be readily adapted to knock out other genes or introduce specific mutations in zebrafish simply by altering the gRNA sequence.
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Highly efficient, genotype-independent transformation and gene editing in watermelon (Citrullus lanatus) using a chimeric ClGRF4-GIF1 gene.
Efficient genetic transformation has the potential to advance research and breeding in watermelon (Citrullus lanatus), but regeneration from tissue culture remains challenging. Previous work showed that expressing a fusion of two interacting transcription factors, GROWTH-REGULATING FACTOR4 (GRF4) and GRF-INTERACTING FACTOR1 (GIF1), improved regeneration in wheat (Triticum aestivum). By overexpressing a chimeric fusion of ClGRF4 and ClGIF1, we achieved highly efficient transformation in watermelon. Mutating the mi396 microRNA target site in ClGRF further boosted the transformation efficiency up to 67.27% in a genotype-independent manner. ClGRF4-GIF1 can also be combined with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing tools to achieve highly efficient gene editing in watermelon, which we used to successfully create diploid seedless watermelon. This research thus puts forward a powerful transformation tool for future watermelon research and breeding.
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Off-target predictions in CRISPR-Cas9 gene editing using deep learning.
MOTIVATION: The prediction of off-target mutations in CRISPR-Cas9 is a hot topic due to its relevance to gene editing research. Existing prediction methods have been developed; however, most of them just calculated scores based on mismatches to the guide sequence in CRISPR-Cas9. Therefore, the existing prediction methods are unable to scale and improve their performance with the rapid expansion of experimental data in CRISPR-Cas9. Moreover, the existing methods still cannot satisfy enough precision in off-target predictions for gene editing at the clinical level. RESULTS: To address it, we design and implement two algorithms using deep neural networks to predict off-target mutations in CRISPR-Cas9 gene editing (i.e. deep convolutional neural network and deep feedforward neural network). The models were trained and tested on the recently released off-target dataset, CRISPOR dataset, for performance benchmark. Another off-target dataset identified by GUIDE-seq was adopted for additional evaluation. We demonstrate that convolutional neural network achieves the best performance on CRISPOR dataset, yielding an average classification area under the ROC curve (AUC) of 97.2% under stratified 5-fold cross-validation. Interestingly, the deep feedforward neural network can also be competitive at the average AUC of 97.0% under the same setting. We compare the two deep neural network models with the state-of-the-art off-target prediction methods (i.e. CFD, MIT, CROP-IT, and CCTop) and three traditional machine learning models (i.e. random forest, gradient boosting trees, and logistic regression) on both datasets in terms of AUC values, demonstrating the competitive edges of the proposed algorithms. Additional analyses are conducted to investigate the underlying reasons from different perspectives. AVAILABILITY AND IMPLEMENTATION: The example code are available at https://github.com/MichaelLinn/off_target_prediction. The related datasets are available at https://github.com/MichaelLinn/off_target_prediction/tree/master/data.
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Advanced molecular therapies for neurological diseases: focus on stroke, alzheimer's disease, and parkinson's disease.
Neurological diseases (NDs) are one of the leading causes of disability and the second leading cause of death globally. Among these stroke, Alzheimer's disease (AD), and Parkinson's disease (PD) are the most common NDs. A rise in the absolute number of individuals affected with these diseases indicates that the current treatment strategies in management and prevention of these debilitating diseases are not effective sufficiently. Therefore, novel treatment strategies are being explored to cure these diseases by addressing the causative mechanisms at the molecular level. Advanced therapies like gene therapy (gene editing and gene silencing) and stem cell therapies aim to cure diseases by gene editing, gene silencing and tissue regeneration, respectively. Gene editing results in the deletion of the aberrant gene or insertion of the corrected gene which can be executed using the CRISPR/Cas gene editing tool a promising treatment strategy being explored for many other prevalent diseases. Gene silencing using siRNA silences the gene by inhibiting protein translation, thereby silencing its expression. Stem cell therapy aims to regenerate damaged cells or tissues because of their ability to divide into any type of cell in the human body. Among these approaches, gene editing and gene silencing have currently been applied in vitro and to animal models, while stem cell therapy has reached the clinical trial stage for the treatment of NDs. The current status of these strategies suggests a promising outcome in their clinical translation.
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CRISPR/Cas9-targeted mutagenesis of the PpTCP4 gene increased tiller number in Poa pratensis.
Poa pratensis, commonly known as Kentucky bluegrass, is a widely used cool-season grass species for turf in lawns and recreational areas worldwide. However, the lack of genome editing platforms has impeded research into enhancing forage crops suitable for land unsuitable for other crops. In this study, we successfully established an Agrobacterium-mediated genetic transformation system for P. pratensis. Furthermore, we successfully produced PpTCP4-edited plants using the CRISPR/Cas9 system. This research represents a significant advancement in the genetic transformation and gene editing of P. pratensis, providing a foundational technology for future investigations into gene function and breeding in P. pratensis.
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Potential of Genome Editing to Improve Aquaculture Breeding and Production.
Aquaculture is the fastest growing food production sector and is rapidly becoming the primary source of seafood for human diets. Selective breeding programs are enabling genetic improvement of production traits, such as disease resistance, but progress is limited by the heritability of the trait and generation interval of the species. New breeding technologies, such as genome editing using CRISPR/Cas9 have the potential to expedite sustainable genetic improvement in aquaculture. Genome editing can rapidly introduce favorable changes to the genome, such as fixing alleles at existing trait loci, creating de novo alleles, or introducing alleles from other strains or species. The high fecundity and external fertilization of most aquaculture species can facilitate genome editing for research and application at a scale that is not possible in farmed terrestrial animals.
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Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants.
The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.
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Polysaccharide-Based Delivery Systems for CRISPR/Cas Gene Therapy: Overcoming Challenges and Advancing Pharmaceutical Solutions.
The advent of CRISPR/Cas gene-editing technology has revolutionized molecular biology, offering unprecedented precision and potential in treating genetic disorders, cancers, and other complex diseases. However, for CRISPR/Cas to be truly effective in clinical settings, one of the most significant challenges lies in the delivery of the CRISPR components, including guide RNA (gRNA) and Cas protein, into specific cells or tissues. Safe, targeted, and efficient delivery remains a critical bottleneck. Viral vectors, lipid nanoparticles, and synthetic polymers have been explored, but they come with limitations, such as immunogenicity, toxicity, and limited delivery capacity. Polysaccharide-based delivery systems, with their natural origin, biocompatibility, and versatile chemical properties, offer a promising alternative that could address these delivery challenges while advancing the pharmaceutical applications of CRISPR/Cas gene therapy.
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