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Synergistic enhancement of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 -mediated gene editing in porcine zygotes through combined lipofection and electroporation of cationic lipid-packaged ribonucleoproteins.
BACKGROUND AND AIM: Genetically engineered pigs are invaluable biomedical models for xenotransplantation and the study of human diseases. Although electroporation (EP) and lipofection are individually effective for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) delivery, their combined application in porcine embryos has not been systematically evaluated. This study aimed to determine whether packaging Cas9-guided RNA complexes in cationic lipids enhances EP-mediated gene editing efficiency without compromising embryonic development. MATERIALS AND METHODS: Porcine zygotes with their zona pellucida removed were edited using RNPs targeting beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2) or growth hormone receptor (GHR) genes. Four treatment groups were tested: (1) EP with RNPs (EP), (2) EP with lipofectamine-packaged RNPs (EPL), (3) transfection with lipofectamine-packaged RNPs before EP (TL + EPL), and (4) EP followed by lipofection (EPL + TL). Blastocyst formation was evaluated morphologically, and mutation rates were assessed by Sanger sequencing followed by tracking of indels by decomposition (TIDE) analysis. RESULTS: Blastocyst formation rates were comparable across all treatments, indicating that lipofectamine packaging and EP caused no detectable cytotoxicity. For B4GALNT2, no mutations were induced by EP alone, whereas TL + EPL treatment significantly increased total and mosaic mutation rates (p < 0.05). For GHR, the total mutation and mosaic mutation rates were likewise higher in TL + EPL compared with EP, although mutation efficiency (indel percentage per edited embryo) remained unchanged. These results suggest that pre-EP lipofection promotes RNP uptake by facilitating lipid-membrane interactions that are potentiated by subsequent membrane destabilization through EP. CONCLUSION: Packaging RNPs in cationic lipids and applying sequential lipofection followed by EP significantly enhances CRISPR/Cas9-mediated mutagenesis in porcine zygotes without affecting developmental competence. This dual-delivery approach provides a simple, reproducible, and low-toxicity workflow for generating gene-edited embryos, with potential applicability to large-animal biomedical models.
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How should the applications of genome editing be assessed and regulated?
An EASAC working group on genome editing recommends that regulators should focus on specific applications of these new techniques rather than attempting to regulate genome editing itself as a new technology.
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In Our Image: The Ethics of CRISPR Genome Editing.
The advent of genome editing technology promises to transform human health, livestock and agriculture, and to eradicate pest species. This transformative power demands urgent scrutiny and resolution of the ethical conflicts attached to the creation and release of engineered genomes. Here, I discuss the ethics surrounding the transformative CRISPR/Cas9-mediated genome editing technology in the contexts of human genome editing to eradicate genetic disease and of gene drive technology to eradicate animal vectors of human disease.
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CRISPR/Cas genome editing improves abiotic and biotic stress tolerance of crops.
Abiotic stress such as cold, drought, saline-alkali stress and biotic stress including disease and insect pest are the main factors that affect plant growth and limit agricultural productivity. In recent years, with the rapid development of molecular biology, genome editing techniques have been widely used in botany and agronomy due to their characteristics of high efficiency, controllable and directional editing. Genome editing techniques have great application potential in breeding resistant varieties. These techniques have achieved remarkable results in resistance breeding of important cereal crops (such as maize, rice, wheat, etc.), vegetable and fruit crops. Among them, CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) provides a guarantee for the stability of crop yield worldwide. In this paper, the development of CRISRR/Cas and its application in different resistance breeding of important crops are reviewed, the advantages and importance of CRISRR/Cas technology in breeding are emphasized, and the possible problems are pointed out.
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An Update on the Application of CRISPR Technology in Clinical Practice.
The CRISPR/Cas system, an innovative gene-editing tool, is emerging as a promising technique for genome modifications. This straightforward technique was created based on the prokaryotic adaptive immune defense mechanism and employed in the studies on human diseases that proved enormous therapeutic potential. A genetically unique patient mutation in the process of gene therapy can be corrected by the CRISPR method to treat diseases that traditional methods were unable to cure. However, introduction of CRISPR/Cas9 into the clinic will be challenging because we still need to improve the technology's effectiveness, precision, and applications. In this review, we first describe the function and applications of the CRISPR-Cas9 system. We next delineate how this technology could be utilized for gene therapy of various human disorders, including cancer and infectious diseases and highlight the promising examples in the field. Finally, we document current challenges and the potential solutions to overcome these obstacles for the effective use of CRISPR-Cas9 in clinical practice.
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Agrobacterium tumefaciens-Mediated Plant Transformation and Gene Editing in Rice.
Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas9-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent most of the rice production in the USA and South America, along with some regions in Asia. This chapter describes a protocol for CRISPR/Cas9 gene editing using Agrobacterium-mediated transformation for the tropical japonica rice cultivar Presidio leading to knock-out mutations in the phytoene desaturase (PDS) gene.
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Strategies to improve homology-based repair outcomes following CRISPR-based gene editing in mosquitoes: lessons in how to keep any repair disruptions local.
Programmable gene editing systems such as CRISPR-Cas have made mosquito genome engineering more practical and accessible, catalyzing the development of cutting-edge genetic methods of disease vector control. This progress, however, has been limited by the low efficiency of homology-directed repair (HDR)-based sequence integration at DNA double-strand breaks (DSBs) and a lack of understanding about DSB repair in mosquitoes. Innovative efforts to optimize HDR sequence integration by inhibiting non-homologous end joining or promoting HDR have been performed in mammalian systems, however many of these approaches have not been applied to mosquitoes. Here, we review some of the most relevant steps of DNA DSB repair choice and highlight promising approaches that influence this choice to enhance HDR in the context of mosquito gene editing.
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Optimizing the delivery of CRISPR/Cas9 ribonucleoproteins for efficient genome editing in bovine embryos.
To assist in establishing a streamlined and efficient workflow for generating gene-edited bovine embryos, we evaluated three transfection approaches for the delivery of CRISPR Cas9-sgRNA ribonucleoproteins into bovine zygotes: lipofection with Lipofectamine CRISPRMAX, and electroporation using either Neon or NEPA21 electroporation systems. Bos taurus prolactin receptor (PRLR) was used as the target gene. The PRLR editing outcomes were analysed by PCR genotyping and Sanger sequencing of individual embryos at day 8 post-fertilization. CRISPRMAX transfection generated up to 30 % PRLR-edited blastocysts (8 % homozygous deletion), without affecting the embryo cleavage (93 %) and blastocyst rate (39 %) relative to non-transfected controls. For both NEPA21 and Neon electroporation, we found that increasing the voltage, length and number of pulses resulted in enhanced gene editing efficiency but compromised embryo cleavage and blastocyst rates. NEPA21 electroporation with a commercial electroporation enhancer reagent produced up to 47.6 % transfected embryos with the PRLR deletion, but with decreased embryo cleavage (62 %) and blastocyst (18 %) rates. Combining NEPA21 electroporation with CRISPRMAX lipofection enhanced the gene editing efficiency to 50 % (23 % homozygous editing), with 64 % embryo cleavage rate and 18 % blastocyst rate. Notably, Neon electroporation resulted in 65.2 % PRLR-edited blastocysts with 21 % homozygous editing (50% cleavage rate and 10 % blastocyst rate). Additional studies may be necessary to further optimize electroporation parameters to achieve an optimal balance between embryo viability and gene editing efficiency. These outcomes will provide valuable insights for improving gene editing workflows for bovines and may help to promote and accelerate the widespread implementation of genome editing technology in livestock.
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Engineered CRISPR-Cas9 nuclease with expanded targeting space.
The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.
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New approaches to moderate CRISPR-Cas9 activity: Addressing issues of cellular uptake and endosomal escape.
CRISPR-Cas9 is rapidly entering molecular biology and biomedicine as a promising gene-editing tool. A unique feature of CRISPR-Cas9 is a single-guide RNA directing a Cas9 nuclease toward its genomic target. Herein, we highlight new approaches for improving cellular uptake and endosomal escape of CRISPR-Cas9. As opposed to other recently published works, this review is focused on non-viral carriers as a means to facilitate the cellular uptake of CRISPR-Cas9 through endocytosis. The majority of non-viral carriers, such as gold nanoparticles, polymer nanoparticles, lipid nanoparticles, and nanoscale zeolitic imidazole frameworks, is developed with a focus toward optimizing the endosomal escape of CRISPR-Cas9 by taking advantage of the acidic environment in the late endosomes. Among the most broadly used methods for in vitro and ex vivo ribonucleotide protein transfection are electroporation and microinjection. Thus, other delivery formats are warranted for in vivo delivery of CRISPR-Cas9. Herein, we specifically revise the use of peptide and nanoparticle-based systems as platforms for CRISPR-Cas9 delivery in vivo. Finally, we highlight future perspectives of the CRISPR-Cas9 gene-editing tool and the prospects of using non-viral vectors to improve its bioavailability and therapeutic potential.
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