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Magnetogenetics with Piezo1 Mechanosensitive Ion Channel for CRISPR Gene Editing.
Regulation of genetic activity in single cells and tissues is pivotal to determine key cellular functions in current biomedicine, yet the conventional biochemical activators lack spatiotemporal precision due to the diffusion-mediated slow kinetics and nonselectivity. Here, we describe a magnetogenetic method for target-specific activation of a clustered regularly interspaced short palindromic repeats (CRISPR) system for the regulation of intracellular proteins. We used magnetomechanical force generated by the magnetic nanostructure to activate pre-encoded Piezo1, the mechanosensitive ion channel, on the target cell. The activated Piezo1 further triggers the intracellular Ca2+ signaling pathway, inducing the pre-encoded genes to express genes of interest (GOIs), which is Cas9 protein for the CRISPR regulation of the target proteins. We demonstrated that this magnetogenetic CRISPR system successfully edits the target genome for both in vitro and pseudo-in vivo environments, providing a versatile magnetic platform for remote gene editing of animals with various size scales.
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Genome editing in fishes and their applications.
There have been revolutionary progresses in genome engineering in the past few years. The newly-emerged genome editing technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats associated with Cas9 (CRISPR/Cas9) have enabled biological scientists to perform efficient and precise targeted genome editing in different species. Fish represent the largest group of vertebrates with many species having values for both scientific research and aquaculture industry. Genome editing technologies have found extensive applications in different fish species for basic functional studies as well asapplied research in such fields as disease modeling and aquaculture. This mini-review focuses on recent advancements and applications of the new generation of genome editing technologies in fish species, with particular emphasis on their applications in understanding reproductive functions because the reproductive axis has been most systematically and best studied among others and its function has been difficult to address with reverse genetics approach.
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Mutagenesis and genome editing in crop improvement: perspectives for the global regulatory landscape.
Plant breeding depends on broad genetic variation. New allelic variation can be produced by targeted or random mutagenesis. Seemingly, random mutagenesis is outdated because clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology is much more precise and potentially faster. Unfortunately, genome editing is not accessible to breeders in many countries due to legal constraints. Therefore, random mutagenesis remains a vital method to create new allelic variation. Mutant offspring, however, suffer from a heavy mutation load, and application in polyploid crops is limited because multiple mutations are typically required. Exploiting random mutations became more efficient due to recent technological advancements, such as sequence-based mutant screening and genomic background selection. In this review, random and targeted mutagenesis will be compared, highlighting the legal situation.
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A simple validation and screening method for CRISPR/Cas9-mediated gene editing in mouse embryos to facilitate genetically modified mice production.
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is a genome engineering method for generating site-specific editing in target genes in a variety of species. It is a common tool for generating mouse models of different diseases. However, detecting target modifications in mouse embryos can be time-consuming and expensive. Accordingly, developing a screening method to confirm gene modification may be useful. We propose herein an evaluation method (cleavage assay - CA) for CRISPR/Cas9-mediated gene editing in preimplantation mouse embryos that allows us to detect mutants efficiently and later on initiate in vivo production without the extensive number of samples needing to be sent for Sanger sequencing and animal usage. Our method is based on the inability of the RNP complex to recognize the target sequence after CRISPR-mediated genome editing due to modification of the target locus. It allows us to establish gene edited mice in a user-friendly fashion with a limited number of mice usage by confirming each step of CRISPR-mediated gene editing of mouse embryos and, therefore, can be considered as a supportive tool to existing procedures for verification of successful CRISPR/Cas9-mediated gene alterations in mouse embryos and further mutant production.
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Enhanced Cas12a multi-gene regulation using a CRISPR array separator.
The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.
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Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish.
The zebrafish (Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish.
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Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs.
The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR-associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing.
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Compact RNA editors with natural miniature Cas13j nucleases.
Clustered regularly interspaced short palindromic repeats-Cas13 effectors are used for RNA editing but the adeno-associated virus (AAV) packaging limitations because of their big sizes hinder their therapeutic application. Here we report the identification of the Cas13j family, with LepCas13j (529 aa) and ChiCas13j (424 aa) being the smallest and most highly efficient variants for RNA interference. The miniaturized Cas13j proteins enable the development of compact RNA base editors. Chi-RESCUE-S, by fusing dChiCas13j with hADAR2dd, demonstrates high efficiency and specificity in A-to-G and C-to-U conversions. Importantly, this system is compatible with single-AAV packaging without the need for protein sequence truncation. It successfully corrected pathogenic mutations, such as APOC3D65N and SCN9AR896Q, to the wild-type forms. In addition, we developed an optimized system, Chi-RESCUE-S-mini3, which pioneered efficient in vivo C-to-U RNA editing of PCSK9 in mice through single-AAV delivery, resulting in reduced total cholesterol levels. These results highlight the potential of Cas13j to treat human diseases.
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CRISPR as agent: a metaphor that rhetorically inhibits the prospects for responsible research.
In 2015, a group of 18 scientists and bioethicists published an editorial in Science calling for "open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome" and recommending that steps be taken to strongly discourage "any attempts at germline genome modification" in humans with this powerful new technology. Press reports compared the essay to a letter written by Paul Berg and 10 other scientists in 1974, also published in Science, calling for a voluntary deferral of certain types of recombinant DNA experimentation. A rhetorical analysis of the metaphors in these two documents, and in the summary statements that came out of the respective National Academy of Sciences conferences they instigated, shows that while they have a lot in common, they are different in at least one important way. The more recent texts deploy conceptual metaphors that portray the biotechnology in question as an autonomous agent, subtly suggesting an inevitability to its development, in contrast to the earlier texts, which portray the scientists who are using the technology as the primary agents who take action. Rhetorical moves depicting biotechnology as an agent in the 2015 texts hint at contemporary skepticism about whether humans can restrain the forward momentum of science and technology in a global context, thus inhibiting scientists from imagining a consequential role for themselves in shaping the future of responsible research.
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A history of genome editing in mammals.
Genome editing is now a routine procedure in many mammalian genetics laboratories. The ostensibly short but intense history of genome-editing approaches illustrates how a disruptive technology can universally colonize a field when this new methodology, conceived to alter mammalian genomes at specific locations, is found to efficiently and robustly deliver results. This review summarizes the early development of genome editing using nucleases, from the pioneering experiments using yeast meganucleases, to the latest prokaryotic nucleases used for precise genome manipulation. Gene-editing nucleases belong to one of three known categories: zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) tools. All operate on the same principle; they are all capable of inducing a double-strand break (DSB) at a defined genomic sequence that is subsequently corrected by endogenous DNA repair mechanisms. DSBs can be repaired through non-homologous end joining (NHEJ), resulting in small insertions and/or deletions (INDELs) and, hence, often leading to gene disruption. Alternatively, DSBs can be repaired through homology-driven repair (HDR), in the presence of donor homologous DNA sequences, resulting in gene-editing events.
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