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In Vivo Investigation of Gene Function in Muscle Stem Cells by CRISPR/Cas9-Mediated Genome Editing.
Skeletal muscle satellite cells (SCs) are adult stem cells responsible for muscle development and injury-induced muscle regeneration. Functional elucidation of intrinsic regulatory factors governing SC activity is constrained partially by the technological limitations in editing SCs in vivo. Although the power of CRISPR/Cas9 in genome manipulation has been widely documented, its application in endogenous SCs remains largely untested. Our recent study generates a muscle-specific genome editing system leveraging the Cre-dependent Cas9 knockin mice and AAV9-mediated sgRNAs delivery, which allows gene disruption in SCs in vivo. Here, we illustrate the step-by-step procedure for achieving efficient editing using the above system.
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The gene-therapy revolution risks stalling if we don't talk about drug pricing.
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A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock-in via CRISPR/Cas9 Technology.
Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery.
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Monkey survives for two years after gene-edited pig-kidney transplant.
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CRISPR-mediated modification of DNA methylation pattern in the new era of cancer therapy.
In the last 2 decades, a wide variety of studies have been conducted on epigenetics and its role in various cancers. A major mechanism of epigenetic regulation is DNA methylation, including aberrant DNA methylation variations such as hypermethylation and hypomethylation in the promoters of critical genes, which are commonly detected in tumors and mark the early stages of cancer development. Therefore, epigenetic therapy has been of special importance in the last decade for cancer treatment. In epigenetic therapy, all efforts are made to modulate gene expression to the normal status. Importantly, recent studies have shown that epigenetic therapy is focusing on the new gene editing technology, CRISPR-Cas9. This tool was found to be able to effectively modulate gene expression and alter almost any sequence in the genome of cells, resulting in events such as a change in acetylation, methylation, or histone modifications. Of note, the CRISPR-Cas9 system can be used for the treatment of cancers caused by epigenetic alterations. The CRISPR-Cas9 system has greater advantages than other available methods, including potent activity, easy design and high velocity as well as the ability to target any DNA or RNA site. In this review, we described epigenetic modulators, which can be used in the CRISPR-Cas9 system, as well as their functions in gene expression alterations that lead to cancer initiation and progression. In addition, we surveyed various species of CRISPR-dead Cas9 (dCas9) systems, a mutant version of Cas9 with no endonuclease activity. Such systems are applicable in epigenetic therapy for gene expression modulation through chemical group editing on nucleosomes and chromatin remodeling, which finally return the cell to the normal status and prevent cancer progression.
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Protocol to rapidly screen CRISPR-Cas9 gene editing outcomes in a cell population by mutating eGFP to a blue or non-fluorescent phenotype.
When designing genome editing therapy, it is crucial to measure outcomes of DNA damage repair. Here, we present a protocol to distinguish the outcome of targeted DNA damage repair from the bottom up, through a previously established readout of enhanced green fluorescent protein (eGFP) to blue fluorescent protein (BFP) mutations. We describe steps for producing eGFP-positive cells and differentiating between non-homologous end joining-induced gene knockout and homology-directed repair-induced-directed mutation in these cells. This protocol has potential for high-throughput and scalable assessment of gene editing techniques. For complete details on the use and execution of this protocol, please refer to Walther et al.1 and Wilbie et al.2.
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Study on the framework of ATP energy cycle system in Escherichia coli.
The high mortality rate associated with single-use CRISPR-Cas9 in Escherichia coli limits its application. Recently, new CRISPR-based techniques for E.coli gene editing have emerged. Research aims to develop a system for rapid, marker-free, multi-site, and multi-copy genome editing in E.coli to advance synthetic biology. ATP, essential for energy in living organisms, plays a crucial role in various metabolic processes. To reduce the cost of ATP-requiring reactions, it is crucial to identify and efficiently express genes in ATP synthesis pathway. This study identified a single ppk gene (No.8) capable of completing the cyclic reaction. Using MUCICAT technology, the ppk gene (No.8) was inserted into various positions and copy numbers in the E.coli genome, resulting in different activity levels. The findings suggest that the difficulty of inserting the ppk gene (No.8) into the genome follows this order: IS186 < 8array < IS186 + 8array < IS1. A single genome insertion can mimic plasmid expression level. This study explores promoter competition and offers solutions, inspiring researchers in constructing the AMP-ATP cycle system in E.coli. KEY POINTS: • The single ppk gene (No.8) can regenerate the AMP-ATP cycle, crucial for ATP-dependent reactions. • Inserting the ppk gene (No.8) into the cr5 site of the E.coli genome achieves expression levels comparable to the pET29a plasmid. • The expression level of the ppk gene (No.8) is not significantly affected by its copy number in the E.coli genome.
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Development of the CRISPR/Cas9 system for genome editing in Riemerella anatipestifer.
Riemerella anatipestifer (R. anatipestifer) is an important pathogen responsible for high mortality rates and severe economic losses in the poultry industry. Research on R. anatipestifer is constrained by limited genetic manipulation tools, highlighting the need for an effective genome editing toolkit. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system from Streptococcus pyogenes has been widely used as a genome editing tool for various bacteria, but has not been used in R. anatipestifer. In this study, we developed a CRISPR/Cas9-based genome editing system, pCasRA-SacB, specifically for R. anatipestifer. This shuttle vector contains the replication origin from R. anatipestifer plasmid pRA0726 and p15A origin for replication in both R. anatipestifer and Escherichia coli (E. coli). It also includes chloramphenicol and cefoxitin resistance genes, which allow for selection in E. coli and R. anatipestifer. The vector features BsaI and SalI sites, enabling the cloning of single guide RNA (sgRNA) and homologous arms. In addition, it contains the high-expression promoter of B739_0921 gene and the promoter of rpsL gene, facilitating the expression of sgRNA and Cas9 protein. Furthermore, the vector features the sucrose-sensitive gene sacB and the oriT region, which enable plasmid curing and the transfer of plasmid via conjugation. Notably, the pCasRA-SacB system enables rapid, efficient, and scarless genome editing in R. anatipestifer, including gene deletion, insertion, and point mutation in the dprA gene, with editing efficiencies of 54.2 %, 100.0 %, and 50.0 %, respectively. In summary, the pCasRA-SacB system not only expands the genome editing toolbox of R. anatipestifer but also is helpful in fundamental research in R. anatipestifer.
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The New Old Colonialism.
The question of how law should regulate the manipulation of the human genome or germline is inflected by the interconnected, intersectional parrying among different systems of moral value. Contract law and constitutional law reflect two poles of interest: the transactional aspects of market valuation and the relational aspects of the web of life that acknowledge "pricelessness." In the decades from the initial decoding of the human genome in 2000 to the emergence of CRISPR technologies, powerful companies and powerful individuals now all but own the fate of our species and the health of our planet. The destructive effects of the realignments we are undergoing are still largely invisible (if not for long) and largely unresponsive to conventional checks.
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Efficient Genome Editing Achieved via Plug-and-Play Adenovirus Piggyback Transport of Cas9/gRNA Complex on Viral Capsid Surface.
The capacity to efficiently deliver the gene-editing enzyme complex to target cells is favored over other forms of gene delivery as it offers one-time hit-and-run gene editing, thus improving precision and safety and reducing potential immunogenicity against edited cells in clinical applications. Here we performed a proof-of-mechanism study and demonstrated that a simian adenoviral vector for DNA delivery can be repurposed as a robust intracellular delivery platform for a functional Cas9/guide RNA (gRNA) complex to recipient cells. In this system, the clinically relevant adenovirus was genetically engineered with a plug-and-display technology based on SpyTag003/SpyCatcher003 coupling chemistry. Under physiological conditions, an off-the-shelf mixture of viral vector with SpyTag003 incorporated into surface capsid proteins and Cas9 fused with SpyCatcher003 led to a rapid titration reaction yielding adenovirus carrying Cas9SpyCatcher003 on the virus surface. The Cas9 fusion protein-conjugated viruses in the presence of a reporter gRNA delivered gene-editing functions to cells with an efficiency comparable to that of a commercial CRISPR/Cas9 transfection reagent. Our data fully validate the adenoviral "piggyback" approach to deliver an intracellularly acting enzyme cargo and, thus, warrant the prospect of engineering tissue-targeted adenovirus carrying Cas9/gRNA for in vivo gene editing.
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