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Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure.
The clustered regularly interspaced short palindromic repeats (CRISPR) system has recently been developed into a powerful genome-editing technology, as it requires only two key components (Cas9 protein and sgRNA) to function and further enables multiplex genome targeting and homology-directed repair (HDR) based precise genome editing in a wide variety of organisms. Here, we report a novel and interesting strategy by using the Drosha-mediated sgRNA-shRNA structure to direct Cas9 for multiplex genome targeting and precise genome editing. For multiplex genome targeting assay, we achieved more than 9% simultaneous mutant efficiency for 3 genomic loci among the puromycin-selected cell clones. By introducing the shRNA against DNA ligase IV gene (LIG4) into the sgRNA-shRNA construct, the HDR-based precise genome editing efficiency was improved as more than 2-fold. Our works provide a useful tool for multiplex and precise genome modifying in mammalian cells.
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Genome Editing Inhibits Retinal Angiogenesis in a Mouse Model of Oxygen-Induced Retinopathy.
This protocol describes a novel approach harnessing the technology of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9-based gene editing for treating retinal angiogenesis. In this system, adeno-associated virus (AAV)-mediated CRISPR/Cas9 was employed to edit the genome of vascular endothelial growth factor receptor (VEGFR)2 in retinal vascular endothelial cells in a mouse model of oxygen-induced retinopathy. The results showed that genome editing of VEGFR2 suppressed pathological retinal angiogenesis. This mouse model mimics a critical aspect of abnormal retinal angiogenesis in patients with neovascular diabetic retinopathy and retinopathy of prematurity, indicating genome editing has high potential for treating angiogenesis-associated retinopathies.
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TALEN or Cas9 - rapid, efficient and specific choices for genome modifications.
Precise modifications of complex genomes at the single nucleotide level have been one of the big goals for scientists working in basic and applied genetics, including biotechnology, drug development, gene therapy and synthetic biology. However, the relevant techniques for making these manipulations in model organisms and human cells have been lagging behind the rapid high throughput studies in the post-genomic era with a bottleneck of low efficiency, time consuming and laborious manipulation, and off-targeting problems. Recent discoveries of TALEs (transcription activator-like effectors) coding system and CRISPR (clusters of regularly interspaced short palindromic repeats) immune system in bacteria have enabled the development of customized TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 to rapidly edit genomic DNA in a variety of cell types, including human cells, and different model organisms at a very high efficiency and specificity. In this review, we first briefly summarize the development and applications of TALENs and CRISPR/Cas9-mediated genome editing technologies; compare the advantages and constraints of each method; particularly, discuss the expected applications of both techniques in the field of site-specific genome modification and stem cell based gene therapy; finally, propose the future directions and perspectives for readers to make the choices.
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Analysis of therapeutic nucleic acids by capillary electrophoresis.
Nucleic acids are getting increased attention to fulfill unmet medical needs. The past five years have seen more than ten FDA approvals of nucleic acid based therapeutics. New analytical challenges have been posed in discovery, characterization, quality control and bioanalysis of therapeutic nucleic acids. Capillary electrophoresis (CE) has proven to be an efficient separation technique and has been widely used for analyzing oligonucleotides and nucleic acids. This review discusses the recent technical advances of CE in nucleic acid analysis such as polymeric matrices, separation conditions and detection methods, and the applications of CE to various therapeutic nucleic acids including antisense oligonucleotide (ASO), small interfering ribonucleic acid (siRNA), messenger RNA (mRNA), gene editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR)-based gene and cell therapy, and other nucleic acid related therapeutics.
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Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system.
CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications, not only for single gene targeting, but also for multiple targeted mutagenesis. To make the most of the multiplexity of CRISPR/Cas9, we established a system for constructing all-in-one expression vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette. We further demonstrated successful examples of multiple targeting including chromosomal deletions in human cells using the all-in-one CRISPR/Cas9 vectors constructed with our novel system. Our system provides an efficient targeting strategy for multiplex genome/epigenome editing, simultaneous activation/repression of multiple genes, and beyond.
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Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes.
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Gene Editing on Center Stage.
Smithies et al. (1985) and Jasin and colleagues (1994) provided proof of concept that homologous recombination (HR) could be applied to the treatment of human disease and that its efficiency could be improved by the induction of double-strand breaks (DSBs). A key advance was the discovery of engineered nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like (TAL) effector nucleases (TALENs), that can generate site-specific DSBs. The democratization and widespread use of genome editing was enabled by the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease system. While genome editing using ZFNs and TALENs has already reached clinical trials, the pace at which genome editing enters human trials is bound to accelerate in the next several years with multiple promising preclinical studies heralding cures for monogenic diseases that are currently difficult to manage or even incurable. Here we review recent advances and current limitations and discuss the path forward using genome editing to understand, treat, and cure genetic diseases.
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CRISPR and iPSCs: Recent Developments and Future Perspectives in Neurodegenerative Disease Modelling, Research, and Therapeutics.
Neurodegenerative diseases are prominent causes of pain, suffering, and death worldwide. Traditional approaches modelling neurodegenerative diseases are deficient, and therefore, improved strategies that effectively recapitulate the pathophysiological conditions of neurodegenerative diseases are the need of the hour. The generation of human-induced pluripotent stem cells (iPSCs) has transformed our ability to model neurodegenerative diseases in vitro and provide an unlimited source of cells (including desired neuronal cell types) for cell replacement therapy. Recently, CRISPR/Cas9-based genome editing has also been gaining popularity because of the flexibility they provide to generate and ablate disease phenotypes. In addition, the recent advancements in CRISPR/Cas9 technology enables researchers to seamlessly target and introduce precise modifications in the genomic DNA of different human cell lines, including iPSCs. CRISPR-iPSC-based disease modelling, therefore, allows scientists to recapitulate the pathological aspects of most neurodegenerative processes and investigate the role of pathological gene variants in healthy non-patient cell lines. This review outlines how iPSCs, CRISPR/Cas9, and CRISPR-iPSC-based approaches accelerate research on neurodegenerative diseases and take us closer to a cure for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, and so forth.
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Gene Editing in Polyploid Crops: Wheat, Camelina, Canola, Potato, Cotton, Peanut, Sugar Cane, and Citrus.
Polyploid crops make up a significant portion of the major food and fiber crops of the world and include wheat, potato, cotton, apple, peanut, citrus, and brassica oilseeds such as rape, canola, and Camelina. The presence of three sets of chromosomes in triploids, four sets in tetraploids, and six sets in hexaploids present significant challenges to conventional plant breeding and, potentially, to efficient use of rapidly emerging gene and genome-editing systems such as zinc finger nucleases, single-stranded oligonucleotides, TALE effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). However, recent studies with each of these techniques in several polyploid crops have demonstrated facile editing of some or all of the genes targeted for modification on homeologous chromosomes. These modifications have allowed improvements in food nutrition, seed oil composition, disease resistance, weed protection, plant breeding procedures, and food safety. Plants and plant products exhibiting useful new traits created through gene editing but lacking foreign DNA may face reduced regulatory restrictions. Such plants can be obtained either by simply selecting for null segregants that have lost their editing transgenes during plant breeding or, even more attractively, by delivery of biodegradable Cas9/sgRNA ribonucleoprotein complexes (i.e., no DNA) into plant cells where they are expressed only transiently but allow for efficient gene editing-a system that has been recently demonstrated in at least two polyploid crops. Such systems that create precise mutations but leave no transgene footprint hold potential promise for assisting with the elimination or great diminution of regulatory processes that presently burden approvals of conventional transgenic crops.
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Development and application of CRISPR/Cas9 to improve anthocyanin pigmentation in plants: Opportunities and perspectives.
Since its discovery in 2012, the novel technology of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has greatly contributed to revolutionizing molecular biology. It has been demonstrated to be an effective approach for identifying gene function and improving some important traits. Anthocyanins are secondary metabolites responsible for a wide spectrum of aesthetic coloration in various plant organs and are beneficial for health. As such, increasing anthocyanin content in plants, especially the edible tissue and organs, is always a main goal for plant breeding. Recently, CRISPR/Cas9 technology has been highly desired to enhance the amount of anthocyanin in vegetables, fruits, cereals, and other attractive plants with more precision. Here we reviewed the recent knowledge concerning CRISPR/Cas9-mediated anthocyanin enhancement in plants. In addition, we addressed the future avenues of promising potential target genes that could be helpful for achieving the same goal using CRISPR/Cas9 in several plants. Thus, molecular biologists, genetic engineers, agricultural scientists, plant geneticists, and physiologists may benefit from CRISPR technology to boost the biosynthesis and accumulation of anthocyanins in fresh fruits, vegetables, grains, roots, and ornamental plants.
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