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Efficient viral delivery of Cas9 into human safe harbor.
Gene editing using CRISPR/Cas9 is a promising method to cure many human genetic diseases. We have developed an efficient system to deliver Cas9 into the adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor, using lentivirus and AAV viral vectors, as a step toward future in vivo transduction. First, we introduced Cas9v1 (derived from Streptococcus pyogenes) at random into the genome using a lentiviral vector. Cas9v1 activity was used when the N-terminal 1.9 kb, and C-terminal 2.3 kb fragments of another Cas9v2 (human codon-optimized) were employed sequentially with specific single-guide RNAs (sgRNAs) and homology donors carried by AAV vectors into the AAVS1 locus. Then, Cas9v1 was removed from the genome by another AAV vector containing sgRNA targeting the long terminal repeat of the lentivirus vector. The reconstituted Cas9v2 in the AAVS1 locus was functional and gene editing was efficient.
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Pervasive donor DNA integration defies precision gene editing of hematopoietic stem cells.
Therapeutic gene editing based on homology-directed repair (HDR) promises to precisely recombine donor template sequences at programmed double-strand break sites. In this issue, Ferrari et al. report that commonly used AAV donors blunt hematopoietic repopulation and result in frequent genomic integrations that could interfere with normal gene expression.
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Targeted editing of tomato carotenoid isomerase reveals the role of 5' UTR region in gene expression regulation.
A deletion created by CRISPR/Cas9 system in the 5' UTR of the carotenoid isomerase gene in tomato leads to downregulation of the gene resulting in the low conversion of prolycopene to lycopene. CRISPR/Cas9 based genome editing is an effective and useful tool adopted from the bacterial immune response system for altering specific, pre-determined DNA sequences in eukaryotes. Such targeted changes are finding wide application in human health as well as in precision breeding of crop plants for improved traits. Mutations in the coding and regulatory regions can have varying impacts on the function of the gene. In the current study, we demonstrate this on tomato carotenoid isomerase, a key gene in the carotenoid biosynthesis pathway. Mutations were generated in the 5' UTR and exon 1 of the carotenoid isomerase gene using CRISPR/Cas9 expression via Agrobacterium-mediated transformation of tomato variety Periyakulam 1 (PKM1). Molecular and biochemical studies demonstrate that CRISPR-mediated point mutations in the exon sequence lead to complete knockout of protein function whereas deletion in 5' UTR region lowers the expression of the gene leading to changes in plant phenotype.
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Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening.
Cas12a is a next-generation gene editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.
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Potential of Gene Editing and Induced Pluripotent Stem Cells (iPSCs) in Treatment of Retinal Diseases.
The advent of gene editing has introduced the ability to make changes to the genome of cells, thus allowing for correction of genetic mutations in patients with monogenic diseases. Retinal diseases are particularly suitable for the application of this new technology because many retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and Leber congenital amaurosis (LCA), are monogenic. Moreover, gene delivery techniques such as the use of adeno-associated virus (AAV) vectors have been optimized for intraocular use, and phase III trials are well underway to treat LCA, a severe form of inherited retinal degeneration, with gene therapy. This review focuses on the use of gene editing techniques and another relatively recent advent, induced pluripotent stem cells (iPSCs), and their potential for the study and treatment of retinal disease. Investment in these technologies, including overcoming challenges such as off-target mutations and low transplanted cell integration, may allow for future treatment of many debilitating inherited retinal diseases.
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Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model.
The prokaryotic CRISPR/Cas9 system has recently emerged as a powerful tool for genome editing in mammalian cells with the potential to bring curative therapies to patients with genetic diseases. However, efficient in vivo delivery of this genome editing machinery and indeed the very feasibility of using these techniques in vivo remain challenging for most tissue types. Here, we show that nonreplicable Cas9/sgRNA ribonucleoproteins can be used to correct genetic defects in skin stem cells of postnatal recessive dystrophic epidermolysis bullosa (RDEB) mice. We developed a method to locally deliver Cas9/sgRNA ribonucleoproteins into the skin of postnatal mice. This method results in rapid gene editing in epidermal stem cells. Using this method, we show that Cas9/sgRNA ribonucleoproteins efficiently excise exon80, which covers the point mutation in our RDEB mouse model, and thus restores the correct localization of the collagen VII protein in vivo. The skin blistering phenotype is also significantly ameliorated after treatment. This study provides an in vivo gene correction strategy using ribonucleoproteins as curative treatment for genetic diseases in skin and potentially in other somatic tissues.
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Generating waxy rice starch with target type of amylopectin fine structure and gelatinization temperature by waxy gene editing.
Waxy rice, which lacks amylose, is an important variant in rice, and its starches have been widely used. New waxy rice varieties generated via the CRISPR/Cas9 gene-editing system is described. Herein, four waxy rice starches with different physicochemical properties were successfully obtained by the CRISPR/Cas9 editing Waxy (Wx) gene. The results showed that the amylose content (AC) of wx mutant starches ranged from 0.26 to 1.78 %, and CZBwx1 starches had the best gel consistency and highest water solubility among all wx mutants. Mutations of Wxb allele produced more short-chains (degree of chain polymerization (DP) 6-11), and less medium- and long-chains (DP12-70) than that of Wxa, while the AC of Wxa allele mutants was higher than the mutations of Wxb allele. The gelatinization temperature (GT) of wxa mutant starches was higher than that of wxb mutant starches. Moreover, we found that the GT and amylopectin fine structure type of waxy rice starch did not change after Wx gene editing. Based on these findings, it is possible to produce waxy rice starch with different physicochemical properties, containing target GT and chain length distributions of amylopectin.
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A new breakthrough in genome editing: the story of Casgevy.
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A platform for precise quantification of gene editing products based on microfluidic chip-based digital PCR.
The new generation of gene editing technologies, primarily based on CRISPR/Cas9 and its derivatives, allows for more precise editing of organisms. However, when the editing efficiency is low, only a small fraction of gene fragments is edited, leaving behind minimal traces and making it difficult to detect and evaluate the editing effects. Although a series of technologies and methods have been developed, they lack the ability for precise quantification and quantitative analysis of these products. Digital polymerase chain reaction (dPCR) offers advantages such as high precision and sensitivity, making it suitable for absolute quantification of nucleic acid samples. In the present study, we developed a novel platform for precise quantification of gene editing products based on microfluidic chip-based dPCR. The results indicated that our assay accurately identified different types of edited samples within a variety of different types, including more complex genomic crops such as tetraploid rapeseed and soybean (highly repetitive sequence). The sensitivity of this detection platform was as low as 8.14 copies per μL, with a detection limit of 0.1%. These results demonstrated the superior performance of the platform, including high sensitivity, low detection limit, and wide applicability, enabling precise quantification and assessment of gene editing efficiency. In conclusion, microfluidic chip-based dPCR was used as a powerful tool for precise quantification and assessment of gene editing products.
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Heparin Specifically Inhibits CRISPR/Cas12 Activation, Enabling Ultrasensitive Heparin Detection and Gene Editing Regulation.
Heparin is a highly sulfated linear glycosaminoglycan that is used as an anticoagulant to prevent and treat thrombotic diseases. Herein, we find that heparin specifically inhibits the activation of the Cas12 protein through the competitive binding of heparin and crRNA to Cas12. Studies illustrate that heparin's high molecular weight and strong negative charge are critical parameters for its inhibitory effect. This unexpected finding was engineered for the detection of heparin, affording a low detection limit of 0.36 ng/mL for fluorometric quantification. We further developed a rapid lateral flow-based system named HepaStrip (heparin strip), allowing heparin monitoring in clinical samples within 20 min. Finally, in vivo investigations revealed that heparin can regulate gene editing with the clusters of the regularly spaced short palindromic repeat (CRISPR)/Cas12 system in Escherichia coli. Heparin blocks the formation of Cas12-crRNA ribonucleoprotein, allowing the application of CRISPR for rapid and field-deployable detection of heparin and unleashing the potential use of heparin in future anti-CRISPR applications.
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