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A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.
The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii.
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Regulation of Gene Expression Using dCas9-SunTag Platforms.
Regulating gene expression is important for basic research and therapeutic applications. The epigenome is a record of genetic modifications such as DNA methylation and histone modifications, and epigenetic changes can play a key role in modifying gene expression. With the advent of genome editing technologies, it has become possible to manipulate the epigenome of specific genomic regions to control gene expression. In particular, CRISPR-Cas9 systems have been used widely for epigenome editing due to their high efficiency, versatility, specificity, and ease of use. Here, we describe a protocol for the upregulation of specific genes using the dCas9-SunTag system.
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[A new toolbox for genome editing].
A new approach, based on the use of peptide nucleic acid molecules to form triple helices and promote locus-specific recombination, demonstrates potential for in vivo gene correction at clinically significant levels and may provide a promising avenue for gene therapy.
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CRISPR-Cas9-Mediated Gene Knockout in a Non-Model Sea Urchin, Heliocidaris crassispina.
Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. Heliocidaris crassispina is an important marine fishery resource with edible gonads. Although H. crassispina has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to H. crassispina. In this study, we targeted genes encoding ETS transcription factor (HcEts) and pigmentation-related polyketide synthase (HcPks1). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When Ets gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the Pks1 gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the Pks1 gene in Hemicentrotus pulcherrimus, mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, Pks1 knockout H. crassispina exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin H. crassispina.
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Biotechnological challenges: The scope of genome editing.
CRISPR/Cas9 can be considered as the biotechnological breakthrough of the century. Genome editing technologies have developed in a vertiginous way. While the genome editing of species, including animals, plants and bacteria has become a commonly used method, the application of CRISPR-Cas9 in human embryos has led to debates and interdisciplinary discussions. This brings multiple challenges for both scientists and those who must regulate the use of these techniques.
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A tool for more specific DNA integration.
The efficiency of targeted DNA insertion by CRISPR transposons is improved.
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Development of CRISPR/Cas Delivery Systems for In Vivo Precision Genome Editing.
Clustered, regularly interspaced, short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) is emerging as a powerful genome-editing tool, enabling precise and targeted modifications of virtually any genomic sequence in living cells. These technologies have potential therapeutic applications for cancers, metabolic diseases, and genetic disorders. However, several major challenges hinder the full realization of their potential. Specifically, CRISPR-Cas9 gene editors, whether delivered as plasmid DNA, mRNA/sgRNA, or ribonucleoprotein (RNP), exhibit poor membrane permeability, restricting their access to the intracellular genome, where the editing occurs. Additionally, these editors lack tissue or organ specificity, raising concerns about off-target editing at the tissue level that causes unwanted genotoxicity. Though a range of delivery carriers has been developed to deliver Cas9 editors, their effectiveness is often limited by a number of barriers at both the extracellular and intracellular levels. Moreover, the prolonged activity of Cas9 increases the risk of off-target editing at the genomic level. Therefore, it is crucial to develop efficient delivery vectors, along with molecular switches to safely regulate Cas9 activity.In this Account, we summarize our recent achievements in developing different types of materials that can efficiently deliver the plasmid DNA encoding Cas9 protein and single-guide RNA (sgRNA), or Cas9 RNP into cells to highlight the design considerations of carriers for safe and efficient delivery in vitro and in vivo. After elucidating the chemical and physical factors that are responsible for encapsulating and delivering these biomacromolecules, we further elucidate how we design the biodegradable polymeric carriers using dynamic disulfide chemistry, emphasize their safe and efficient delivery features for genome-editing biomacromolecules, and also introduce the integration of the intracellular delivery of genome-editing biomacromolecules with microneedle-based transdermal delivery to promote therapeutic genome editing for inflammatory skin disorders. Finally, we review how we exploit optical, chemical, and genetic switches to control the Cas9 activity in conjunction with targeted delivery to address the spatiotemporal specificity of gene editing in vivo and demonstrate their precision therapy against cancer and colitis treatment as proof-of-concept examples. In the final part, we will summarize the progress we have made and propose the future directions that may impact the field based on our own research outcomes.
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Further analysis of barley MORC1 using a highly efficient RNA-guided Cas9 gene-editing system.
Microrchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL-type ATPase and an S5-fold. In plants, MORC proteins were first discovered in a genetic screen for Arabidopsis thaliana mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and genome stabilization. Little is known about the role of MORC proteins of cereals, especially because knockout (KO) mutants were not available and assessment of loss of function relied only on RNAi strategies, which were arguable, given that MORC proteins in itself are influencing gene silencing. Here, we used a Streptococcus pyogenes Cas9 (SpCas9)-mediated KO strategy to functionally study HvMORC1, one of the current seven MORC members of barley. Using a novel barley RNA Pol III-dependent U3 small nuclear RNA (snRNA) promoter to drive expression of the synthetic single guide RNA (sgRNA), we achieved a very high mutation frequency in HvMORC1. High frequencies of mutations were detectable by target sequencing in the callus, the T0 generation (77%) and T1 generation (70%-100%), which constitutes an important improvement of the gene-editing technology in cereals. Corroborating and extending earlier findings, SpCas9-edited hvmorc1-KO barley, in clear contrast to Arabidopsis atmorc1 mutants, had a distinct phenotype of increased disease resistance to fungal pathogens, while morc1 mutants of either plant showed de-repressed expression of transposable elements (TEs), substantiating that plant MORC proteins contribute to genome stabilization in monocotyledonous and dicotyledonous plants.
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Genome editing: the breakthrough technology for inherited retinal disease?
Genetic alterations resulting in a dysfunctional retinal pigment epithelium and/or degenerating photoreceptors cause impaired vision. These juxtaposed cells in the retina of the posterior eye are crucial for the visual cycle or phototransduction. Deficits in these biochemical processes perturb neural processing of images capturing the external environment. Notably, there is a distinct lack of clinically approved pharmacological, cell- or gene-based therapies for inherited retinal disease. Gene editing technologies are rapidly advancing as a realistic therapeutic option. Areas covered: Recent discovery of endonuclease-mediated gene editing technologies has culminated in a surge of investigations into their therapeutic potential. In this review, the authors discuss gene editing technologies and their applicability in treating inherited retinal diseases, the limitations of the technology and the research obstacles to overcome before editing a patient's genome becomes a viable treatment option. Expert opinion: The ability to strategically edit a patient's genome constitutes a treatment revolution. However, concerns remain over the safety and efficacy of either transplanting iPSC-derived retinal cells following ex vivo gene editing, or with direct gene editing in vivo. Ultimately, further refinements to improve efficacy and safety profiles are paramount for gene editing to emerge as a widely available treatment option.
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Mapping the coevolution, leadership and financing of research on viral vectors, RNAi, CRISPR/Cas9 and other genomic editing technologies.
Genomic editing technologies are developing rapidly, promising significant developments for biomedicine, agriculture and other fields. In the present investigation, we analyzed and compared the process of innovation for six genomic technologies: viral vectors, RNAi, TALENs, meganucleases, ZFNs and CRISPR/Cas including the profile of the main research institutions and their funders, to understand how innovation evolved and what institutions influenced research trajectories. A Web of Science search of papers on viral vectors RNAi, CRISPR/Cas, TALENs, ZFNs and meganucleases was used to build a citation network of 16,746 papers. An analysis of network clustering combined with text mining was performed. For viral vectors, a long-term process of incremental innovation was identified, which was largely publicly funded in the United States and the European Union. The trajectory of RNAi research included clusters related to the study of RNAi as a biological phenomenon and its use in functional genomics, biomedicine and pest control. A British philanthropic organization and a US pharmaceutical company played a key role in the development of basic RNAi research and clinical application respectively, in addition to government and academic institutions. In the case of CRISPR/Cas research, basic science discoveries led to the technical improvements, and these two in turn provided the information required for the development of biomedical, agricultural, livestock and industrial applications. The trajectory of CRISPR/Cas research exhibits a geopolitical division of the investigation efforts between the US, as the main producer and funder of basic research and technical improvements, and Chinese research institutions increasingly leading applied research. Our results reflect a change in the model for financing science, with reduced public financing for basic science and applied research on publicly funded technological developments in the US, and the emergence of China as a scientific superpower, with implications for the development of applications of genomic technologies.
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