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Production of genetically engineered mice with higher efficiency, lower mosaicism, and multiplexing capability using maternally expressed Cas9.
The CRISPR/Cas9 system is widely used to generate gene-edited animals. Here, we developed an efficient system for generating genetically modified mice using maternal Cas9 from Cas9 transgenic mice. Using this system, we achieved lower mosaicism and higher rates of knock-in success, gene-editing, and birth compared to the similar parameters obtained using exogenously administered Cas9 (mRNA/protein) system. Furthermore, we successfully induced simultaneous mutations at multiple loci (a maximum of nine). Our novel gene-editing system based on maternal Cas9 could potentially facilitate the generation of mice with single and multiple gene modifications.
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Co-delivery of Sorafenib and CRISPR/Cas9 Based on Targeted Core-Shell Hollow Mesoporous Organosilica Nanoparticles for Synergistic HCC Therapy.
The rapid development of CRISPR/Cas9 systems has opened up tantalizing prospects to sensitize cancers to chemotherapy using efficient targeted genome editing, but safety concerns and possible off-target effects of viral vectors remain a major obstacle for clinical application. Thus, the construction of novel nonviral tumor-targeting nanodelivery systems has great potential for the safe application of CRISPR/Cas9 systems for gene-chemo-combination therapy. Here, we report a polyamidoamine-aptamer-coated hollow mesoporous silica nanoparticle for the co-delivery of sorafenib and CRISPR/Cas9. The core-shell nanoparticles had good stability, enabled ultrahigh drug loading, targeted delivery, and controlled-release of the gene-drug combination. The nanocomplex showed >60% EGFR-editing efficiency without off-target effects in all nine similar sites, regulating the EGFR-PI3K-Akt pathway to inhibit angiogenesis, and exhibited a synergistic effect on cell proliferation. Importantly, the co-delivery nanosystem achieved efficient EGFR gene therapy and caused 85% tumor inhibition in a mouse model. Furthermore, the nanocomplex showed high accumulation at the tumor site in vivo and exhibited good safety with no damage to major organs. Due to these properties, the nanocomplex provides a versatile delivery approach for efficient co-loading of gene-drug combinations, allowing for precise gene editing and synergistic inhibition of tumor growth without apparent side effects on normal tissues.
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Establishment of Agrobacterium-mediated genetic transformation and CRISPR/Cas9-guided gene editing in Elymus nutans.
Elymus nutans, an allohexaploid (2n = 6x = 42) species with a StStHHYY genome, is a native perennial in the alpine grasslands of the Qinghai-Xizang Plateau, and has been widely used for artificial pasture and ecological restoration as a forage grass with highest yield on the plateau. Nevertheless, the lack of a stable transformation system has impeded further efforts to trait improvement of E. nutans. In the present study, we established a reliable Agrobacterium-mediated genetic transformation system for E. nutans, and successfully generated EnTCP4-edited plants using the CRISPR/Cas9 system. The editing efficiency achieved 19.23 % in E. nutans. Knocking out EnTCP4 significantly delayed flowering and enhanced water-deficit stress resistance. This research represents a significant breakthrough in the genetic transformation and gene editing of E. nutans, laying a technological foundation to gain insight into gene functions and molecular breeding in E. nutans.
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CRISPR/Cas9 genome editing demonstrates functionality of the autoimmunity-associated SNP rs12946510.
Genome-wide association studies (GWAS) map genetic associations of complex traits with precision limited to a linkage disequilibrium group. To translate GWAS results into new understanding of disease mechanisms, individual causative polymorphisms and their target genes should be identified. CRISPR/Cas9 genome editing can be used to create isogenic cell lines bearing alternative genotypes of candidate single-nucleotide polymorphisms to test their causality and to reveal gene targets. An intergenic polymorphism rs12946510 is associated with multiple sclerosis, inflammatory bowel disease and asthma. We created sublines of the T-helper cell line bearing alternative genotypes of rs12946510 and showed that its risk ("T") allele is associated with lower expression of IKZF3 and ORMDL3 genes and reduced cell activation. Our editing procedure can become an effective tool for discovering new genes involved in pathogenesis of complex diseases.
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CRISPR/Cas9 Genome Editing of Human-Induced Pluripotent Stem Cells Followed by Granulocytic Differentiation.
Research on patient-derived induced pluripotent stem cells (iPSCs) could immensely benefit from the implementation of CRISPR/Cas9 genome editing of iPSCs, creating unique opportunities such as the establishment of isogenic iPSC lines for disease modeling or personalized patient-specific drug screenings. Here we describe a stepwise protocol of safe, efficient, and selection-free CRISPR/Cas9-mediated gene correction or knockout in human iPSCs followed by 3D spin-embryoid body (EB)-based hematopoietic/neutrophilic iPSC-differentiation.
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'Artificial spermatid'-mediated genome editing†.
For years, extensive efforts have been made to use mammalian sperm as the mediator to generate genetically modified animals; however, the strategy of sperm-mediated gene transfer (SMGT) is unable to produce stable and diversified modifications in descendants. Recently, haploid embryonic stem cells (haESCs) have been successfully derived from haploid embryos carrying the genome of highly specialized gametes, and can stably maintain haploidy (through periodic cell sorting based on DNA quantity) and both self-renewal and pluripotency in long-term cell culture. In particular, haESCs derived from androgenetic haploid blastocysts (AG-haESCs), carrying only the sperm genome, can support the generation of live mice (semi-cloned, SC mice) through oocyte injection. Remarkably, after removal of the imprinted control regions H19-DMR (differentially methylated region of DNA) and IG-DMR in AG-haESCs, the double knockout (DKO)-AG-haESCs can stably produce SC animals with high efficiency, and so can serve as a sperm equivalent. Importantly, DKO-AG-haESCs can be used for multiple rounds of gene modifications in vitro, followed by efficient generation of live and fertile mice with the expected genetic traits. Thus, DKO-AG-haESCs (referred to as 'artificial spermatids') combed with CRISPR-Cas technology can be used as the genetically tractable fertilization agent, to efficiently create genetically modified offspring, and is a versatile genetic tool for in vivo analyses of gene function.
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Delivery of CRISPR/Cas9 system by AAV as vectors for gene therapy.
The adeno-associated virus (AAV) is a defective single-stranded DNA virus with the simplest structure reported to date. It constitutes a capsid protein and single-stranded DNA. With its high transduction efficiency, low immunogenicity, and tissue specificity, it is the most widely used and promising gene therapy vector. The clustered regularly interspaced short palindromic sequence (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system is an emerging technology that utilizes cas9 nuclease to specifically recognize and cleave target genes under the guidance of small guide RNA and realizes gene editing through homologous directional repair and non-homologous recombination repair. In recent years, an increasing number of animal experiments and clinical studies have revealed the great potential of AAV as a vector to deliver the CRISPR/cas9 system for treating genetic diseases and viral infections. However, the immunogenicity, toxicity, low transmission efficiency in brain and ear tissues, packaging size limitations of AAV, and immunogenicity and off-target effects of Cas9 protein pose several clinical challenges. This research reviews the role, challenges, and countermeasures of the AAV-CRISPR/cas9 system in gene therapy.
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Gene therapy for hereditary hearing loss.
Gene therapy is a technique by which exogenous genetic material is introduced into target cells to treat or prevent diseases caused by genetic mutations. Hearing loss is the most common sensory disorder. Genetic factors contribute to approximately 50 % of all cases of profound hearing loss, and more than 150 independent genes have been reported as associated with hearing loss. Recent advances in CRISPR/Cas based gene-editing tools have facilitated the development of gene therapies for hereditary hearing loss (HHL). Viral delivery vectors, and especially adeno-associated virus (AAV) vectors, have been demonstrated as safe and efficient carriers for the delivery of transgenes into inner ear cells in animal models. More importantly, AAV-mediated gene therapy can restore hearing in some children with hereditary deafness. However, there are many different types of HHL that need to be identified and evaluated to determine appropriate gene therapy options. In the present review, we summarize recent animal model-based advances in gene therapy for HHL, as well as gene therapy strategies, gene-editing tools, delivery vectors, and administration routes. We also discuss the strengths and limitations of different gene therapy methods and describe future challenges for the eventual clinical application of gene therapy for HHL.
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An RNAi suppressor activates in planta virus-mediated gene editing.
RNA-guided CRISPR/Cas9 technology has been developed for gene/genome editing (GE) in organisms across kingdoms. However, in planta delivery of the two core GE components, Cas9 and small guide RNA (sgRNA), often involves time-consuming and labor-intensive production of transgenic plants. Here we show that Foxtail mosaic virus, a monocot- and dicot-infecting potexvirus, can simultaneously express Cas9, sgRNA, and an RNAi suppressor to efficiently induce GE in Nicotiana benthamiana through a transgenic plant-free manner.
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Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development.
A standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 forelimb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes.
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