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Genome Modification Technologies and Their Applications in Avian Species.
The rapid development of genome modification technology has provided many great benefits in diverse areas of research and industry. Genome modification technologies have also been actively used in a variety of research areas and fields of industry in avian species. Transgenic technologies such as lentiviral systems and piggyBac transposition have been used to produce transgenic birds for diverse purposes. In recent years, newly developed programmable genome editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) have also been successfully adopted in avian systems with primordial germ cell (PGC)-mediated genome modification. These genome modification technologies are expected to be applied to practical uses beyond system development itself. The technologies could be used to enhance economic traits in poultry such as acquiring a disease resistance or producing functional proteins in eggs. Furthermore, novel avian models of human diseases or embryonic development could also be established for research purposes. In this review, we discuss diverse genome modification technologies used in avian species, and future applications of avian biotechnology.
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NEMO Links Nuclear Factor-κB to Human Diseases.
The nuclear factor (NF)-κB essential modulator (NEMO) is a key regulator in NF-κB-mediated signaling. By transmitting extracellular or intracellular signals, NEMO can control NF-κB-regulated genes. NEMO dysfunction is associated with inherited diseases such as incontinentia pigmenti (IP), ectodermal dysplasia, anhidrotic, with immunodeficiency (EDA-ID), and some cancers. We focus on molecular studies, human case reports, and mouse models emphasizing the significance of NEMO molecular interactions and modifications in health and diseases. This knowledge opens new opportunities to engineer suitable drugs that may putatively target precise NEMO functions attributable to various diseases, while leaving other functions intact, and eliminating cytotoxicity. Indeed, with the advent of novel gene editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9, treating some inherited diseases may in the long run, become a reality.
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Liposome-Based Carriers for CRISPR Genome Editing.
The CRISPR-based genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR), has sparked renewed interest in gene therapy. This interest is accompanied by the development of single-guide RNAs (sgRNAs), which enable the introduction of desired genetic modifications at the targeted site when used alongside the CRISPR components. However, the efficient delivery of CRISPR/Cas remains a challenge. Successful gene editing relies on the development of a delivery strategy that can effectively deliver the CRISPR cargo to the target site. To overcome this obstacle, researchers have extensively explored non-viral, viral, and physical methods for targeted delivery of CRISPR/Cas9 and a guide RNA (gRNA) into cells and tissues. Among those methods, liposomes offer a promising approach to enhance the delivery of CRISPR/Cas and gRNA. Liposomes facilitate endosomal escape and leverage various stimuli such as light, pH, ultrasound, and environmental cues to provide both spatial and temporal control of cargo release. Thus, the combination of the CRISPR-based system with liposome delivery technology enables precise and efficient genetic modifications in cells and tissues. This approach has numerous applications in basic research, biotechnology, and therapeutic interventions. For instance, it can be employed to correct genetic mutations associated with inherited diseases and other disorders or to modify immune cells to enhance their disease-fighting capabilities. In summary, liposome-based CRISPR genome editing provides a valuable tool for achieving precise and efficient genetic modifications. This review discusses future directions and opportunities to further advance this rapidly evolving field.
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iCRISEE: an integrative analysis of CRISPR screen by reducing false positive hits.
Clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) technology has become a popular tool for the study of genome function, and the use of this technology can achieve large-scale screening studies of specific phenotypes. Several analysis tools for CRISPR/Cas9 screening data have been developed, while high false positive rate remains a great challenge. To this end, we developed iCRISEE, an integrative analysis of CRISPR ScrEEn by reducing false positive hits. iCRISEE can dramatically reduce false positive hits and it is robust to different single guide RNA (sgRNA) library by introducing precise data filter and normalization, model selection and valid sgRNA number correction in data preprocessing, sgRNA ranking and gene ranking. Furthermore, a powerful web server has been presented to automatically complete the whole CRISPR/Cas9 screening analysis, where we integrated the main hypothesis in multiple algorithms as a full workflow, including quality control, sgRNA extracting, sgRNA alignment, sgRNA ranking, gene ranking and pathway enrichment. In addition, output of iCRISEE, including result mapping, sample clustering, sgRNA ranking and gene ranking, can be easily visualized and downloaded for publication. Taking together, iCRISEE presents to be the state-of-the-art and user-friendly tool for CRISPR screening data analysis. iCRISEE is available at https://www.icrisee.com.
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Efficient Immune Cell Genome Engineering with Enhanced CRISPR Editing Tools.
Clustered regularly interspaced short palindromic repeats (CRISPR)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of intracellular sensors. Likewise, lymphocytes of the adaptive immune system are difficult to modify using CRISPR-enhanced homology-directed repair because of inefficient or toxic delivery of donor templates using transient transfection methods. To overcome these challenges and limitations, we modified existing tools and developed three alternative methods for CRISPR-based genome editing using a hit-and-run transient expression strategy, together with a convenient system for promoting gene expression using CRISPRa. Overall, our CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering provide efficient alternatives to existing methods and have wide application not only in terms of hematopoietic cells but also other mammalian cell types of interest.
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Targeted long-read sequencing captures CRISPR editing and AAV integration outcomes in brain.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing is an emerging therapeutic modality that shows promise in Huntington's disease and spinocerebellar ataxia (SCA) mouse models. However, advancing CRISPR-based therapies requires methods to fully define in vivo editing outcomes. Here, we use polymerase-free, targeted long-read nanopore sequencing and evaluate single- and dual-gRNA AAV-CRISPR editing of human ATXN2 in transgenic mouse models of SCA type 2 (SCA2). Unbiased high sequencing coverage showed 10%-25% editing. Along with intended edits there was AAV integration, 1%-2% of which contained the entire AAV genome and were largely unmethylated. More than 150 kb deletions at target loci and rearrangements of the transgenic allele (1%) were also found. In contrast, PCR-based nanopore sequencing showed bias for partial AAV fragments and inverted terminal repeats (ITRs) and failed to detect full-length AAV. Cumulatively this work defines the spectrum of outcomes of CRISPR editing in mouse brain after AAV gene transfer using an unbiased long-read sequencing approach.
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Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA.
The CRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR- associated protein 9) system, a highly efficient, simple, and easy genome editing technology, offers significant potential for genetic engineering and has been commonly applied in gene function studies in Drosophila melanogaster. However, when using CRISPR/Cas9 system to edit Drosophila melanogaster gene, Cas9 and sgRNA expression elements exist in different Drosophila melanogaster individuals, and Cas9 and sgRNA must be integrated into an individual through a complex genetic hybridization process, which has a long and complex operation cycle In this study, on the basis of the CRISPR/Cas9 system, we introduced the tRNA-sgRNA system and triplex elements, used triplex elements to link Cas9 and tRNA-sgRNA genes, stabilized the end of Cas9 mRNA after single transcript cutting, and made the expression of both Cas9 protein and sgRNA with a single transcript a reality. And as we obtained the corresponding phenotypic progeny in one hybridization, genetic manipulation was simplified. We found that conditional knockout of the white(w) gene in the Drosophila melanogaster eye and the broad(br) gene in the adult wing disc resulted in corresponding phenotypes that matched expectations using our new conditional gene editing system. So the significant advances in this new conditional gene editing system over the existing CRISPR/Cas9 system are that it is more efficient, extendable, and easy to use.
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Refining Hepatocyte Models to Capture the Impact of CYP2D6*10 Utilizing a PITCh System.
CYP2D6 variants contain various single nucleotide polymorphisms as well as differing levels of metabolic activity. Among these, one of the less active variants CYP2D6*10 (100C > T) is the most prevalent mutation in East Asians, including Japanese. This mutation leads to an amino acid substitution from proline to serine, which reduces the stability of CYP2D6 and consequently decreases its metabolic activity. In this study, we used a genome editing technology called the Precise Integration into Target Chromosome (PITCh) system to stably express six drug-metabolizing enzymes (CYP3A4, POR, uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), CYP1A2, CYP2C19, CYP2C9, and CYP2D6*10) in HepG2 (CYP2D6*10 KI-HepG2) cells to examine the effect of CYP2D6*10 on drug metabolism prediction. The protein expression levels of CYP2D6 in CYP2D6*10 KI-HepG2 cells were reduced relative to those in the CYP3A4-POR-UGT1A1-CYP1A2-CYP2C19-CYP2C9-CYP2D6 knock-in-HepG2 (CYPs-UGT1A1 KI-HepG2) cells. Consistent with the CYP2D6 protein expression results, CYP2D6 metabolic activity in CYP2D6*10 KI-HepG2 cells was reduced relative to CYPs-UGT1A1 KI-HepG2 cells. We successfully generated CYP2D6*10 KI-HepG2 cells with highly expressed, functional CYP2D6*10, as well as CYP1A2, 2C9, 2C19 and 3A4. CYP2D6*10 KI-HepG2 cells could be an invaluable model for hepatic metabolism and hepatotoxicity studies in East Asians, including Japanese.
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High-Efficiency Genome Editing in Arabidopsis Using YAO Promoter-Driven CRISPR/Cas9 System.
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Versatile and multifaceted CRISPR/Cas gene editing tool for plant research.
The ability to create desirable gene variants through targeted changes offers tremendous opportunities for the advancement of basic and applied plant research. Gene editing technologies have opened new avenues to perform such precise gene modifications in diverse biological systems. These technologies use sequence-specific nucleases, such as homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR/Cas) complexes to enable targeted genetic manipulations. Among these, the CRISPR/Cas system has emerged as a broadly applicable and valued gene editing system for its ease of use and versatility. The adaptability of the CRISPR/Cas system has facilitated rapid and continuous innovative developments to the precision and applications of this technology, since its introduction less than a decade ago. Although developed in animal systems, the simple and elegant CRISPR/Cas gene editing technology has quickly been embraced by plant researchers. From early demonstration in model plants, the CRISPR/Cas system has been successfully adapted for various crop species and enabled targeting of agronomically important traits. Although the approach faces several efficiency and delivery related challenges, especially in recalcitrant crop species, continuous advances in the CRISPR/Cas system to address these limitations are being made. In this review, we discuss the CRISPR/Cas technology, its myriad applications and their prospects for crop improvement.
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