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Nonviral Ex Vivo Genome Editing in Mouse Bona Fide Hematopoietic Stem Cells with CRISPR/Cas9.
Knock-in therapy, in which an insertion site can be controlled, would be more suitable for the treatment of genetic blood disorders as compared to conventional gene therapy with lentivirus vectors that introduce genes into the genome randomly. Recent advancements in genome editing technology have substantially improved the knock-in efficiency, making it a reality. We present the details of a virus-free CRISPR/Cas9-based genome editing method for bona fide mouse hematopoietic stem cells.
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High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome.
Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50-100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.
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Comparison of Multiple Strategies for Precision Transgene Knock-In in Gallus gallus Genome via Microhomology-Mediated End Joining.
Precision exogenous gene knock-in is an attractive field for transgenic Gallus gallus (chicken) generation. In this article, we constructed multiple Precise Integration into Target Chromosome (PITCh) plasmid systems mediated by microhomology-mediated end-joining (MMEJ) for large-fragment integration in DF-1 cells and further assess the possibility of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a genomic safe harbor for chickens. We designed three targeted sgRNAs for the all-in-one plasmid at the 3'UTR of GAPDH near the stop codon. The donor-plasmid-carrying microhomology arms correspond to sgRNA and EGFP fragments in the forward and reverse directions. MMEJ-mediated EGFP insertion can be efficiently expressed in DF-1 cells. Moreover, the differences between the forward and reverse fragments indicated that promoter interference does affect the transfection efficiency of plasmids and cell proliferation. The comparison of the 20 bp and 40 bp microhomology arms declared that the short one has higher knock-in efficiency. Even though all three different transgene insertion sites in GAPDH could be used to integrate the foreign gene, we noticed that the G2-20R-EGFP cell reduced the expression of GAPDH, and the G3-20R-EGFP cell exhibited significant growth retardation. Taken together, G1, located at the 3'UTR of GAPDH on the outer side of the last base of the terminator, can be a candidate genomic safe harbor (GSH) loci for the chicken genome. In addition, deleted-in-azoospermia-like (DAZL) and actin beta (ACTB) site-specific gene knock-in indicated that MMEJ has broad applicability and high-precision knock-in efficiency for genetically engineered chickens.
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Potential of small extracellular vesicles as Cas9 delivery tool: a promising approach for gene editing livestock gametes and embryos.
Genome editing is a rapidly advancing technology with transformative potential in livestock, offering opportunities that range from enhanced production traits to the generation of biomedical models for human disease and xenotransplantation. The CRISPR/Cas9 system, originally identified as a bacterial defense mechanism, has become the most widely used tool for precise genome editing. In this review, we first summarize the potential applications of CRISPR/Cas9 in livestock and highlight notable successes to date. We then address the ongoing challenges associated with delivering CRISPR/Cas9 into gametes and embryos, as current methods such as microinjection and electroporation often result in high mosaicism and cellular damage. We subsequently introduce extracellular vesicles (EVs) as a promising alternative delivery system. Secreted by virtually all cell types, EVs can efficiently transport bioactive molecules and are readily internalized by gametes and embryos. Although EV-mediated delivery of CRISPR/Cas9 has shown success in somatic cells, its use in reproductive cells remains largely unexplored. We review emerging strategies for loading EVs with CRISPR/Cas components and discuss the potential advantages of combining this approach with recently developed smaller Cas variants to overcome delivery barriers. Collectively, these innovations support the promise of EVs as a biologically compatible, efficient, and minimally invasive system for targeted genome editing in livestock reproduction.
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Genome editing in the green alga Chlamydomonas: past, present practice and future prospects.
The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.
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Genetic Modification of Mice Using Prime Editing.
Genetically modifying mice traditionally involved complex methods of designing and validating targeting constructs, embryonic stem cell electroporation and selection, blastocyst injection, and breeding chimeras for germline transmission. Such arduous steps were best carried out by specialized gene targeting cores in academia or through expensive commercial vendors. Further, the time from initiation to completion of a project often took at least 1 year and, in some cases, much longer (or never), with no guarantees of success. The RNA-programmable CRISPR system of gene editing has greatly streamlined the generation of gene modifications (e.g., small substitutions, insertions, and deletions) in the mouse with high rates of success. Several editing platforms exist for gene/genome targeting in mice and other animal models previously difficult or impossible to alter. Here, we provide a simplified method of generating genetically modified mice using the prime editing platform. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design, cloning, and synthesis of engineered pegRNA (epegRNA) Basic Protocol 2: Microinjection of PE2 components into mouse zygote Basic Protocol 3: Genotyping founder mice and breeding for germline transmission.
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Applications of CRISPR-Cas9 in gynecological cancer research.
Gynecological cancers pose a significant threat to women's health worldwide, with cervical cancer, ovarian cancer, and endometrial cancer having high incidences. Current gynecological cancer treatment methods mainly include surgery, chemotherapy, radiotherapy, and chemoradiotherapy. The CRISPR-Cas9 gene editing technology as a new therapeutic method has shown tremendous effect in the treatment of other cancers, promoting research on its potential therapeutic effect in gynecological cancer. In this article, we reviewed the current research status of CRISPR-Cas9 technology in gynecological cancer, focusing on the importance of studying the mechanism of CRISPR-Cas9 in gynecological cancer treatment, thereby laying a foundation for further research on its clinical application.
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Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.
The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.
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CRISPR/Cas9-mediated gene editing of GmJAGGED1 increased yield in the low-latitude soybean variety Huachun 6.
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An update on CRISPR-Cas12 as a versatile tool in genome editing.
Gene editing techniques, which help in modification of any DNA sequence at ease, have revolutionized the world of Genetic engineering. Although there are other gene-editing techniques, CRISPR has emerged as the chief and most preferred tool due to its simplicity and capacity to execute effective gene editing in a wide range of organisms. Although Cas9 has widely been employed for genetic modification over the years, Cas12 systems have lately emerged as a viable option. This review primarily focuses on assessing Cas12-mediated mutagenesis and elucidating the editing efficacy of both Cpf1 (Cas12a) and C2c1 (Cas12b) systems in microbes, plants, and other species. Also, we reviewed several genetic alterations that have been performed with these Cas12 systems to improve editing efficiency. Furthermore, the experimental benefits and applications of Cas12 systems are highlighted in this study.
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