Dataset Information

Near-infrared upconversion-activated CRISPR-Cas9 system: A remote-controlled gene editing platform.

ABSTRACT: As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light-responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as "nanotransducers" that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9. In addition, by preparing a single guide RNA targeting a tumor gene (polo-like kinase-1), our strategies have successfully inhibited the proliferation of tumor cell via NIR light-activated gene editing both in vitro and in vivo. Overall, this exogenously controlled method presents enormous potential for targeted gene editing in deep tissues and treatment of a myriad of diseases.

SUBMITTER: Pan Y

PROVIDER: S-EPMC6447385 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Near-infrared upconversion-activated CRISPR-Cas9 system: A remote-controlled gene editing platform.

Pan Yongchun Y Yang Jingjing J Luan Xiaowei X Liu Xinli X Li Xueqing X Yang Jian J Huang Ting T Sun Lu L Wang Yuzhen Y Lin Youhui Y Song Yujun Y

Science advances 20190403 4

As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light-responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as "nanotransduce ...[more]

PMID: 30949579

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Project description:UnlabelledThe clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3'?5' exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters.ImportanceRecent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways.

Project description:Babesia bovis, the most virulent causative agent of bovine babesiosis, is prevalent in tropical and subtropical regions of the world. Although the whole-genome sequence was released more than a decade ago, functional analysis of the genomics of this parasite is hampered by the limited breadth of genetic engineering tools. In this study, we implemented the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system for B. bovis and demonstrated its potential for genome editing. Cas9 and human dihydrofolate reductase (hDHFR) were simultaneously expressed by the B. boviselongation factor-1α bidirectional promoter, and a single guide RNA was expressed via the B. bovisU6 spliceosomal RNA promoter. Using a single plasmid construct, we were able to add an epitope tag to spherical body protein 3 (SBP3), introduce a point mutation into thioredoxin peroxidase 1 (tpx-1) to impair the function of the product, and replace the tpx-1 open reading frame with the other protein. Epitope tagging of SBP3 was efficient using this system, with a negligible number of remaining wild-type parasites and a pure transgenic population produced by allelic replacement of tpx-1 This advancement in genetic engineering tools for B. bovis will aid functional analysis of the genome and underpin characterization of candidate drug and vaccine targets.IMPORTANCEBabesia bovis is the most virulent cause of bovine babesiosis worldwide. The disease consequences are death, abortion, and economical loss due to reduced milk and meat production. Available vaccines are not effective, treatment options are limited, and emergence of drug and acaricide resistance has been reported from different regions. There is an urgent need to identify new drug and vaccine targets. Greater than half of the genes in B. bovis genome, including several expanded gene families which are unique for Babesia spp., have no predicted function. The available genetic engineering tools are based on conventional homologous recombination, which is time-consuming and inefficient. In this study, we adapted the CRISPR/Cas9 system as a robust genetic engineering tool for B. bovis This advancement will aid future functional studies of uncharacterized genes.

Dataset Information

Near-infrared upconversion-activated CRISPR-Cas9 system: A remote-controlled gene editing platform.

Publications

Near-infrared upconversion-activated CRISPR-Cas9 system: A remote-controlled gene editing platform.

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