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Genome editing of Spp1 by inhalable CRISPR/Cas9 formulation for treating pulmonary fibrosis.
Secreted phosphoprotein 1 (Spp1) encoding osteopontin (OPN), a matrix cell protein with pro-inflammatory and pro-necrotic tissue properties, plays a crucial role in the onset and progression of idiopathic pulmonary fibrosis (IPF). In order to treat IPF by taking advantage of Spp1, we herein developed an inhalable system composed of calcium phosphate/ poly (lactic-co-glycolic acid) (PLGA) core-shell nanoparticles which are loaded with CRISPR/Cas9 system targeting Spp1 to investigate its therapeutic potential. Specifically, the plasmid encoding Cas9 and single-guide RNA (sgRNA) selectively targeting Spp1 gene was first condensed by calcium phosphate to form Cas9 complexes, which was then encapsulated by PLGA to formulate into a gene-editing inhalable delivery system (termed CaP/Cas9/PLGA). Interestingly, the aerosolized inhaled delivery of CaP/Cas9/PLGA nanoparticles results in the effective traverse of mucosal barriers to fibrotic lungs, where they are internalized by lung cells without inducing noticeable cytotoxicity. Following endo/lysosomal escape and gene expression of CRISPR system, the disruption of Spp1 gene by Cas9/sgRNA induces the mutation frequency exceeding 30 %, resulting in efficient down-regulation of OPN level. In a bleomycin-induced pulmonary fibrosis mouse model, the inhalation of aerosolized CaP/Cas9/PLGA complexes significantly attenuates fibrosis development and improves lung function with undetectable systemic toxicity. This current study defines an innovative inhalable gene-editing formulation and offers a promising gene therapy modality for treating IPF.
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CRISPR/Cpf1-mediated mutagenesis and gene deletion in industrial filamentous fungi Aspergillus oryzae and Aspergillus sojae.
In industrial applications such as fermentation and heterologous protein production, various Aspergillus oryzae and A. sojae strains are used. Although genetic engineering techniques have been developed for these filamentous fungi, applying such classical techniques to many strains is difficult. Therefore, the establishment of innovative technologies applicable to various industrial strains is required. We previously developed a genome editing technology using the CRISPR/Cas9 system for the efficient genetic engineering of A. oryzae; however, this system is limited by its protospacer adjacent motif sequence. In A. sojae, no genetic engineering using genome editing has been developed. In this study, we aimed to develop a genome editing technology using the Cpf1 nuclease for the genetic engineering of A. oryzae and A. sojae. AMA1-based genome editing vectors bearing codon-optimized cpf1 expression cassettes were constructed, and guide RNA expression cassettes were inserted into the Cpf1 genome editing vectors. Using the resultant plasmids, we performed mutagenesis of the AowA and sC genes in A. oryzae and the AswA gene in A. sojae. We deleted these genes by co-introducing the Cpf1 genome editing plasmid and the donor plasmid. Our study demonstrates that the CRISPR/Cpf1 system can be used as an efficient alternative to the CRISPR/Cas9 system to genetically engineer A. oryzae and as a new approach for efficient genetic engineering of A. sojae.
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Strategies of Bladder Reconstruction after Partial or Radical Cystectomy for Bladder Cancer.
The standard strategy is to reconstruct bladder by use of bowel segments as material in bladder cancer with radical cystectomy clinically. Both natural derived and non natural derived materials are investigated in bladder reconstruction. Studies on mechanical bladder, bladder transplantation and bladder xenotransplantation are currently limited although heart and kidney transplantation or xenotransplantation are successful to a certain extent, and bone prostheses are applied in clinical contexts. Earlier limited number of studies associated with bladder xenograft from animals to humans were not particular promising in results. Although there have been investigations on pig to human cardiac xenotransplantation with CRISPR Cas9 gene editing, the CRISPR Cas technique is not yet widely researched in porcine bladder related gene editing for the potential of human bladder replacement for bladder cancer. The advancement of technologies such as gene editing, bioprinting and induced pluripotent stem cells allow further research into partial or whole bladder replacement strategies. Porcine bladder is suggested as a potential source material for bladder reconstruction due to its alikeness to human bladder. Challenges that exist with all these approaches need to be overcome. This paper aims to review gene editing technology such as the CRISPR Cas systems as tools in bladder reconstruction, bladder xenotransplantation and hybrid bladder with technologies of induced pluripotent stem cells and genome editing, bioprinting for bladder replacement for bladder reconstruction and to restore normal bladder control function after cystectomy for bladder cancer.
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CRISPR-Cas9 in acute myeloid leukaemia: Current state-of-art and future perspectives.
CRISPR-Cas9 gene editing technology has gained attention as a new, reliable and manageable tool for the treatment of previously incurable monogenic diseases. Besides exciting results in this setting, ethical, safety and crucial technical issues have not been fully clarified. More importantly, the role of this potent editing tool in the context of a genetically complex and heterogeneous hematologic malignancy such as acute myeloid leukemia (AML) has not yet been defined to date. In this review we aim to summarize and exploring the ultimate CRISPR-cas9 based strategies for diagnosis, risk stratification and treatment in the context of AML.
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Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets.
Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.
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High-throughput methods for genome editing: the more the better.
During the last decade, targeted genome-editing technologies, especially clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) technologies, have permitted efficient targeting of genomes, thereby modifying these genomes to offer tremendous opportunities for deciphering gene function and engineering beneficial traits in many biological systems. As a powerful genome-editing tool, the CRISPR/Cas systems, combined with the development of next-generation sequencing and many other high-throughput techniques, have thus been quickly developed into a high-throughput engineering strategy in animals and plants. Therefore, here, we review recent advances in using high-throughput genome-editing technologies in animals and plants, such as the high-throughput design of targeted guide RNA (gRNA), construction of large-scale pooled gRNA, and high-throughput genome-editing libraries, high-throughput detection of editing events, and high-throughput supervision of genome-editing products. Moreover, we outline perspectives for future applications, ranging from medication using gene therapy to crop improvement using high-throughput genome-editing technologies.
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Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology.
Barley is among four of the most important cereal crops with respect to global production. Increasing barley yields to desired levels can be achieved by the genetic manipulation of cytokinin content. Cytokinins are plant hormones that regulate many developmental processes and have a strong influence on grain yield. Cytokinin homeostasis is regulated by members of several multigene families. CKX genes encode the cytokinin oxidase/dehydrogenase enzyme, which catalyzes the irreversible degradation of cytokinin. Several recent studies have demonstrated that the RNAi-based silencing of CKX genes leads to increased grain yields in some crop species. To assess the possibility of increasing the grain yield of barley by knocking out CKX genes, we used an RNA-guided Cas9 system to generate ckx1 and ckx3 mutant lines with knockout mutations in the HvCKX1 and HvCKX3 genes, respectively. Homozygous, transgene-free mutant lines were subsequently selected and analyzed. A significant decrease in CKX enzyme activity was observed in the spikes of the ckx1 lines, while in the ckx3 lines, the activity remained at a similar level to that in the control plants. Despite these differences, no changes in grain yield were observed in either mutant line. In turn, differences in CKX activity in the roots between the ckx1 and ckx3 mutants were reflected via root morphology. The decreased CKX activity in the ckx1 lines corresponded to greater root length, increased surface area, and greater numbers of root hairs, while the increased CKX activity in the ckx3 mutants gave the opposite results. RNA-seq analysis of the spike and root transcriptomes revealed an altered regulation of genes controlling cytokinin metabolism and signaling, as well as other genes that are important during seed development, such as those that encode nutrient transporters. The observed changes suggest that the knockout of a single CKX gene in barley may be not sufficient for disrupting cytokinin homeostasis or increasing grain yields.
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Visualization of Chromatin Dynamics by Live Cell Microscopy Using CRISPR/Cas9 Gene Editing and ANCHOR Labeling.
The organization of the eukaryotic nucleus facilitates functional chromatin contacts which regulate gene transcription. Despite this being extensively studied through population-based chromatin contact mapping and microscopic observations in single cells, the spatiotemporal dynamics of chromatin behavior have largely remained elusive. The current methods to label and observe specific endogenous genomic loci in living cells have been challenging to implement and too invasive to biological processes. In this protocol, we describe the use of a recently developed DNA labelling strategy (ANCHOR) with CRISPR/Cas9 gene editing, to discreetly label genes for live cell imaging to study chromatin dynamics. Our approach improves on some of the fundamental shortfalls associated with current labelling strategies and has the potential for multiplexed observations.
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Optimized Guide RNA Selection Improves Streptococcus pyogenes Cas9 Gene Editing of Human Hematopoietic Stem and Progenitor Cells.
Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research because the method is fast, viral free, and shows fewest off target effects. We evaluated various parameters to genetically engineer human hematopoietic stem and progenitor cells (HSPCs) using Streptococcus pyogenes Cas9 (spCas9) RNPs, and achieve gene editing efficiencies up to 80%. We find that guide RNA (gRNA) design is critical to achieve high gene editing efficiencies. However, finding effective gRNAs for HSPCs can be challenging, while the contribution of numerous in silico models is unclear. By screening more than 120 gRNAs, our data demonstrate that in silico gRNA prediction models are ineffective. In this study, we established a time- and cost-efficient in vitro transcribed gRNA screening model in K562 cells that predicts effective gRNAs for HSPCs. RNP based screening thus outperforms in silico modeling and we report that gene editing is equally efficient in distinct CD34+ HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation, or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.
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Csy4-based vector system enables conditional chimeric gene editing in zebrafish without interrupting embryogenesis.
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