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Found: 26 079
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Realigning gene editing with clinical research ethics: What the "CRISPR Twins" debacle means for Chinese and international research ethics governance.
The announcement of the "CRISPR babies" reignited the debate surrounding the ethical, legal and social implications of germline gene editing. Despite having been conducted in the context of a clinical trial, Dr. Jiankui He's research appears to have violated both Chinese regulations and standard ethical procedures, as well as internationally accepted research and bioethical standards. It is within this context that our commentary surrounding the question of the enforceability of Chinese regulations in such a case. We argue that Chinese regulations do align with internationally accepted standards. Yet, the question remains, in what ways can China strengthen and update its regulatory framework to better address the benefits and challenges associated with emerging technologies, delineate clear enforcement mechanisms and specify criteria for ethics approval.
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Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR.
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[Basics and applications of genome editing technology].
Genome editing with programmable site-specific nucleases is an emerging technology that enables the manipulation of targeted genes in many organisms and cell lines. Since the development of the CRISPR-Cas9 system in 2012, genome editing has rapidly become an indispensable technology for all life science researchers, applicable in various fields. In this seminar, we will introduce the basics of genome editing and focus on the recent development of genome editing tools and technologies for the modification of various organisms and discuss future directions of the genome editing research field, from basic to medical applications.
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CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art.
High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S. cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.
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Engineering a Future with VCA: Applying Genetic Circuits to Engineer Tissues for Vascularized Composite Allotransplantation.
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CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding.
Presence of the integrated endogenous banana streak virus (eBSV) in the B genome of plantain (AAB) is a major challenge for breeding and dissemination of hybrids. As the eBSV activates into infectious viral particles under stress, the progenitor Musa balbisiana and its derivants, having at least one B genome, cannot be used as parents for crop improvement. Here, we report a strategy to inactivate the eBSV by editing the virus sequences. The regenerated genome-edited events of Gonja Manjaya showed mutations in the targeted sites with the potential to prevent proper transcription or/and translational into functional viral proteins. Seventy-five percent of the edited events remained asymptomatic in comparison to the non-edited control plants under water stress conditions, confirming inactivation of eBSV into infectious viral particles. This study paves the way for the improvement of B genome germplasm and its use in breeding programs to produce hybrids that can be globally disseminated.
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CRISPR/Cas9 Guide RNA Design Rules for Predicting Activity.
A critical stage in performing gene editing experiments using the CRISPR/Cas9 system is the design of guide RNA (gRNA). In this chapter, we conduct a review of the current gRNA design rules for maximizing on-target Cas9 activity while minimizing off-target activity. In addition, we present some of the currently available computational tools for gRNA activity prediction and assay design.
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CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells.
Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 (FANCD1) primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was employed to target and correct a FANCD1 gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase). FANCD1 function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.
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A Golden Gate and Gateway double-compatible vector system for high throughput functional analysis of genes.
A major research topic nowadays is to study and understand the functions of the increasing number of predicted genes that have been discovered through the complete genome sequencing of many plant species. With the aim of developing tools for rapid and convenient gene function analysis, we have developed a set of "pGate" vectors based on the principle of Golden gate and Gateway cloning approaches. These vectors combine the positive aspects of both Golden gate and Gateway cloning strategies. pGate vectors can not only be used as Golden gate recipient vectors to assemble multiple DNA fragments in a pre-defined order, but they can also work as an entry vector to transfer the assembled DNA fragment(s) to a large number of already-existing, functionally diverse, Gateway compatible destination vectors without adding additional nucleotides during cloning. We show the pGate vectors are effective and convenient in several major aspects of gene function analyses, including BiFC (Bimolecular fluorescence complementation) to analyze protein-protein interaction, amiRNA (artificial microRNA) candidate screening and as assembly of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats, CRISPR-associated protein-9 nuclease) system elements together for genome editing. The pGate system is a practical and flexible tool which can facilitate plant gene function research.
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Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology.
One of the goals of synthetic biology is to build regulatory circuits that control cell behavior, for both basic research purposes and biomedical applications. The ability to build transcriptional regulatory devices depends on the availability of programmable, sequence-specific, and effective synthetic transcription factors (TFs). The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR) system, recently harnessed for transcriptional regulation in various heterologous host cells, offers unprecedented ease in designing synthetic TFs. We review how CRISPR can be used to build synthetic gene circuits and discuss recent advances in CRISPR-mediated gene regulation that offer the potential to build increasingly complex, programmable, and efficient gene circuits in the future.
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