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Direct Parental (DIPA) CRISPR in the jewel wasp, Nasonia vitripennis.
While clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology has demonstrated remarkable promise as a gene-editing tool, its application in certain insects, such as the jewel wasp, Nasonia vitripennis, has been hindered by a lack of a tractable method for reagent delivery. Direct Parental (DIPA-) CRISPR recently emerged as a facile way to induce gene lesions because it involves adult injection with commercially available Cas9-sgRNA with no helper reagent. However, DIPA-CRISPR has so far been tested in only a few insects. Here, we have assessed the amenability of DIPA-CRISPR in N. vitripennis by targeting two eye pigmentation genes, cinnabar and vermilion, which function in the ommochrome pathway. Successful generation of lesions in both genes demonstrated the functionality of DIPA-CRISPR in N. vitripennis and its potential application to other genes, thereby expanding the range of insects suitable for this method. We varied two parameters, Cas9-sgRNA concentration and injection volume, to determine optimal injection conditions. We found that the larger injection volume coupled with either higher or lower reagent concentration was needed for consistent mutation production. However, DIPA-CRISPR yields an overall low mutation rate in N. vitripennis when compared to other tested insects, a characteristic that may be attributed to a proportionally low vitellogenic import efficiency in the jewel wasp. We discuss different factors that may be considered in determining when DIPA-CRISPR may be preferable over other reagent delivery methods.
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Frontiers in CRISPR.
CRISPR-based approaches to genetic engineering are progressing at a rapid pace and present exciting new avenues for science, medicine, and technology. Many of the most cutting-edge advances in genome engineering are encompassed in the Research Articles, Reviews, and Perspectives in this special issue, often with an eye toward future directions for the field. Yet, many questions remain at this new frontier. We asked over 100 CRISPR researchers, including our contributing authors, for their perspectives on some of the most pressing questions surrounding the future of genome engineering and the CRISPR-Cas platform, the challenges that lie ahead, and opportunities for chemists and chemical biologists to drive creative molecular solutions.
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Advances in large-scale DNA engineering with the CRISPR system.
In recent years, DNA engineering technology has undergone significant advancements, with clustered regularly interspaced short palindromic repeats (CRISPR)-based target-specific DNA insertion emerging as one of the most rapidly expanding and widely studied approaches. Traditional DNA insertion technologies employing recombinases typically involve introducing foreign DNA into genes in vivo by either pre-engineering recognition sequences specific to the recombinase or through genetic crossing to incorporate the requisite recognition sequence into the target gene. However, CRISPR-based gene insertion technologies have advanced to streamline this engineering process by combining the CRISPR-Cas module with recombinase enzymes. This process enables accurate and efficient one-step insertion of foreign DNA into the target gene in vivo. Here we provide an overview of the latest developments in CRISPR-based gene insertion technologies and discusses their potential future applications.
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CRISPR and Gene Editing: A Game-changer in Drug Development.
CRISPR and gene editing technologies have emerged as transformative tools in medicine, offering unprecedented precision in targeting genetic disorders and revolutionizing drug development. This review explores the multifaceted impact of CRISPR across various medical domains, from hereditary diseases to infectious diseases and cancer. The potential of CRISPR in personalized medicine, therapeutic innovation, and pandemic prevention is highlighted, along with its role in reshaping traditional drug development processes. However, alongside its promise, ethical considerations loom large, particularly regarding germline editing and equitable access to treatments. The commercialization of CRISPR poses further challenges, raising questions about affordability and healthcare equity. Collaboration among scientists, policymakers, and the public is emphasized to navigate the ethical and societal implications of CRISPR responsibly. As the field advances, it is essential to ensure that the benefits of CRISPR are realized while addressing potential risks and maintaining a commitment to the well-being of future generations.
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CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.
Numerous feasible methods for inserting large fragments of exogenous DNA sequences into the zebrafish genome have been developed, as has genome editing technology using programmable nucleases. However, the coding sequences of targeted endogenous genes are disrupted, and the expression patterns of inserted exogenous genes cannot completely recapitulate those of endogenous genes. Here we describe the establishment of a novel strategy for endogenous promoter-driven and microhomology-mediated end-joining-dependent integration of a donor vector using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9. We successfully integrated mCherry into the final coding sequence of targeted genes to generate seamless transgenic zebrafish lines with high efficiency. This novel seamless transgenesis technique not only maintained the integrity of the endogenous gene but also did not disrupt the function of targeted gene. Therefore, our microhomology-mediated end-joining-mediated transgenesis strategy may have broader applications in gene therapy. Moreover, this novel seamless gene-editing strategy in zebrafish provides a valuable new transgenesis technique, which was driven by endogenous promoters and in vivo animal reporter modes for translational medicine. It is expected to be a standard gene-editing technique in the field of zebrafish, leading to some important breakthroughs for studies in early embryogenesis.-Luo, J.-J., Bian, W.-P., Liu, Y., Huang, H.-Y., Yin, Q., Yang, X.-J., Pei, D.-S. CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.
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Advancing CRISPR-Based Programmable Platforms beyond Genome Editing in Mammalian Cells.
Human diseases are caused by dysregulation of cellular biological programs that are encoded in DNA. Unveiling the endogenous programs and encoding new programs into the genome are key to creating novel diagnostic and therapeutic strategies. CRISPR/Cas9, originally identified in bacteria, has revolutionized genome editing in mammalian cells. Recent advances in CRISPR technologies have provided new programmable platforms for modifying cell function and behavior. CRISPR-based transcriptional regulators and modified gRNAs have enabled multiplexed regulation and visualization of genome dynamics with spatiotemporal precision. Using these toolkits, genome-scale screening platforms can identify key genetic elements or combinations thereof that modulate phenotypes in mammalian cells. In addition, imaging platforms for multiplexed genomic labeling have been created to study the conformation and dynamics of chromatin in living cells, which are essential for genome function. Furthermore, CRISPR-based computation and memory platforms have been built in living mammalian cells by using DNA as a data processing and storage medium to regulate and monitor cellular behaviors. The conditional regulation of CRISPR-based parts has enabled the design of complex multilayered biological programs. CRISPR-based memory platforms can continuously record biological events as mutations in defined DNA loci. By making use of base editors, CRISPR-based computation and memory platforms have been interconnected to perform logic operations based on past events. These technologies open up new avenues for understanding biological phenomena and designing mammalian cells as living machines for biomedical applications.
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CRISPR-induced double-strand breaks trigger recombination between homologous chromosome arms.
CRISPR-Cas9-based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms-often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.
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Establishment of a CRISPR-Cas9-Mediated Genome Editing System in Flax.
Flax is an important crop used for oil and fiber production. Although genetic engineering has been possible in flax, it is not commonly used to produce cultivars. However, the use of genome editing technology, which can produce site-specific mutations without introducing foreign genes, may be a valuable tool for creating elite cultivars that can be easily cultivated. The purpose of this study was to investigate the potential of genome editing in flax by establishing the clustered regularly interspaced short palindromic repeats (CR ISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) genome editing system using the phytoene desaturase (PDS) gene, which produces albino mutants that are easily identifiable. Four sgRNAs were designed from two PDS genes of Flax (LuPDS1 and LuPDS2), and CRISPR-Cas9 genome editing vectors were constructed. After gene transformation, albino phenotypes were observed in transformed callus and regenerated plantlets on selection media. Polymerase chain reaction (PCR) amplification and sequencing of the PDS genes revealed deletions and insertions in the albino tissues, indicating successful editing of the PDS genes. Potential off-target sites were analyzed, but no off-target mutations were found, indicating the specificity of the CRISPR-Cas9 system. The establishment of a flax genome editing system using the CRISPR-Cas9 technology opens up new possibilities for the genetic engineering of flax. This study demonstrates the potential of genome editing in creating elite cultivars that can be easily cultivated, which can have significant implications for the flax industry.
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Chemical engineering of CRISPR-Cas systems for therapeutic application.
Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed molecular biology and the future of gene-targeted therapeutics. CRISPR systems comprise a CRISPR-associated (Cas) endonuclease and a guide RNA (gRNA) that can be programmed to guide sequence-specific binding, cleavage, or modification of complementary DNA or RNA. However, the application of CRISPR-based therapeutics is challenged by factors such as molecular size, prokaryotic or phage origins, and an essential gRNA cofactor requirement, which impact efficacy, delivery and safety. This Review focuses on chemical modification and engineering approaches for gRNAs to enhance or enable CRISPR-based therapeutics, emphasizing Cas9 and Cas12a as therapeutic paradigms. Issues that chemically modified gRNAs seek to address, including drug delivery, physiological stability, editing efficiency and off-target effects, as well as challenges that remain, are discussed.
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CRISPR/Cas Systems as Diagnostic and Potential Therapeutic Tools for Enterohemorrhagic Escherichia coli.
Following its discovery as an adaptive immune system in prokaryotes, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been developed into a multifaceted genome editing tool. This review compiles findings aimed at implementation of this technology for selective elimination or attenuation of enterohemorrhagic Escherichia coli (EHEC). EHEC are important zoonotic foodborne pathogens that cause hemorrhagic colitis and can progress to the life-threatening hemolytic uremic syndrome (HUS). Advancements in the application of CRISPR methodology include laboratory detection and identification of EHEC, genotyping, screening for pathogenic potential, and engineering probiotics to reduce microbial shedding by cattle, the primary source of human infection. Genetically engineered phages or conjugative plasmids have been designed to target and inactivate genes whose products are critical for EHEC virulence.
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