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Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9.
The revolutionary RNA-guided endonuclease CRISPR/Cas9 system has proven to be a powerful tool for gene editing in a plethora of organisms. Here, utilizing this system we developed an efficient protocol for the generation of heritable germline mutations in the parasitoid jewel wasp, Nasonia vitripennis, a rising insect model organism for the study of evolution, development of axis pattern formation, venom production, haplo-diploid sex determination, and host-symbiont interactions. To establish CRISPR-directed gene editing in N. vitripennis, we targeted a conserved eye pigmentation gene cinnabar, generating several independent heritable germline mutations in this gene. Briefly, to generate these mutants, we developed a protocol to efficiently collect N. vitripennis eggs from a parasitized flesh fly pupa, Sarcophaga bullata, inject these eggs with Cas9/guide RNA mixtures, and transfer injected eggs back into the host to continue development. We also describe a flow for screening mutants and establishing stable mutant strains through genetic crosses. Overall, our results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in N. vitripennis, with strong potential for expansion to target critical genes, thus allowing for the investigation of several important biological phenomena in this organism.
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A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.
Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.
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Identification of a Rice Leaf Width Gene Narrow Leaf 22 (NAL22) through Genome-Wide Association Study and Gene Editing Technology.
Rice leaf width (RLW) is a crucial determinant of photosynthetic area. Despite the discovery of several genes controlling RLW, the underlying genetic architecture remains unclear. In order to better understand RLW, this study conducted a genome-wide association study (GWAS) on 351 accessions from the rice diversity population II (RDP-II). The results revealed 12 loci associated with leaf width (LALW). In LALW4, we identified one gene, Narrow Leaf 22 (NAL22), whose polymorphisms and expression levels were associated with RLW variation. Knocking out this gene in Zhonghua11, using CRISPR/Cas9 gene editing technology, resulted in a short and narrow leaf phenotype. However, seed width remained unchanged. Additionally, we discovered that the vein width and expression levels of genes associated with cell division were suppressed in nal22 mutants. Gibberellin (GA) was also found to negatively regulate NAL22 expression and impact RLW. In summary, we dissected the genetic architecture of RLW and identified a gene, NAL22, which provides new loci for further RLW studies and a target gene for leaf shape design in modern rice breeding.
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Review on Advancement of AI in Synthetic Biology.
The way biological systems are built and designed has been revolutionized by synthetic biology. Further enhancements like predictive modeling, optimization and systematic design of complex biological systems, is now possible due to integration of Artificial Intelligence into synthetic biology. This review shares insights on the role of AI in advancement of synthetic biology, including genome editing, metabolic pathway optimization and biological circuit design etc. AI-driven tools contribute to the increased efficiency and precision. Application of deep learning and machine learning has made it possible to make CRISPR-cas9, de novo protein design and gene circuit development more precise. However, there are still some persistent challenges, especially in curating high-quality biological datasets and bridging interdisciplinary gaps between computational and experimental scientists. Future perspectives focus on causal reasoning in AI models, integration of physics based algorithms, and promoting collaboration across disciplines to achieve breakthroughs in both synthetic biology and AI. By joining these fields, the transformative power of synthetic biology and AI can be unlocked and applied in the fields of medicine, biotechnology and environmental sustainability, pioneering a way for a new era of bioengineering.
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CRISPR/Cas9-Based Antiviral Strategy: Current Status and the Potential Challenge.
From its unexpected discovery as a bacterial adaptive immune system to its countless applications as one of the most versatile gene-editing tools, the CRISPR/Cas9 system has revolutionized every field of life science. Virology is no exception to this ever-growing list of CRISPR/Cas9-based applications. Direct manipulation of a virus genome by CRISPR/Cas9 has enabled a systematic study of cis-elements and trans-elements encoded in a virus genome. In addition, this virus genome-specific mutagenesis by CRISPR/Cas9 was further funneled into the development of a novel class of antiviral therapy targeting many incurable chronic viral infections. In this review, a general concept on the CRISPR/Cas9-based antiviral strategy will be described first. To understand the current status of the CRISPR/Cas9-based antiviral approach, a series of recently published antiviral studies involving CRISPR/Cas9-mediated control of several clinically-relevant viruses including human immunodeficiency virus, hepatitis B virus, herpesviruses, human papillomavirus, and other viruses will be presented. Lastly, the potential challenge and future prospect for successful clinical translation of this CRISPR/Cas9-based antiviral method will be discussed.
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A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes.
The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful-focusing on CRISPRi as a potential application-as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species.IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.
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Engineering extracellular vesicles to deliver CRISPR ribonucleoprotein for gene editing.
Clustered regularly interspaced palindromic repeats (CRISPR) is a gene editing tool with tremendous therapeutic potential. Recently, ribonucleoprotein (RNP) complex-based CRISPR systems have gained momentum due to their reduction of off-target editing. This has coincided with the emergence of extracellular vesicles (EVs) as a therapeutic delivery vehicle due to its low immunogenicity and high capacity for manipulation. EVs are cell-derived membranous nanoparticles which mediate the intercellular transfer of molecular components. Current technologies achieve CRISPR RNP encapsulation into EVs through EVs biogenesis, thereby avoiding unnecessary physical, chemical or biological manipulations to the vesicles directly. Herein, we identify sixteen EVs-based CRISPR RNP encapsulation strategies, each with distinct genetic features to encapsulate CRISPR RNP. According to the molecular mechanism facilitating the encapsulation process, there are six strategies of encapsulating Cas9 RNP into virus-like particles based on genetic fusion, seven into EVs based on protein tethering, and three based on sgRNA-coupled encapsulation. Additionally, the incorporation of a targeting moiety to the EVs membrane surface through EVs biogenesis confers tropism and increases delivery efficiency to specific cell types. The targeting moieties include viral envelope proteins, recombinant proteins containing a ligand peptide, single-chain fragment variable (scFv) antibodies, and integrins. However, current strategies still have a number of limitations which prevent their use in clinical trials. Among those, the incorporation of viral proteins for encapsulation of Cas9 RNP have raised issues of biocompatibility due to host immune response. Future studies should focus on genetically engineering the EVs without viral proteins, enhancing EVs delivery specificity, and promoting EVs-based homology directed repair. Nevertheless, the integration of CRISPR RNP encapsulation and tropism technologies will provide strategies for the EVs-based delivery of CRISPR RNP in gene therapy and disease treatment.
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The PROTECTOR strategy employs dCas orthologs to sterically shield off-target sites from CRISPR/Cas activity.
Off-target mutagenesis of CRISPR/Cas systems must be solved to facilitate safe gene therapy. Here, we report a novel approach, termed "PROTECTOR", to shield known off-target sites by directing the binding of an orthologous nuclease-dead Cas protein to the off-target site to sterically interfere with Cas activity. We show that this method reduces off-target mutation rates of two well-studied guide RNAs without compromising on-target activity and that it can be used in combination with high-fidelity Cas enzymes to further reduce off-target editing. This expands the suite of off-target mitigation strategies and offers an ability to protect off-target sites even when their sequences are fully identical to target sites.
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Huntington's chorea: emerging fields in therapeutics (Review).
Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disorder characterized by motor dysfunction, cognitive decline, and psychiatric disturbances. It is caused by CAG repeat expansions in the HTT gene, resulting in the formation of mutant huntingtin protein that aggregates and disrupts neuronal function. This review outlines the pathogenesis of HD, including genetic, molecular, and environmental factors. Additionally, current management approaches and emerging therapeutic strategies-such as RNA interference, antisense oligonucleotides (ASOs), peptide inhibitors, and CRISPR/Cas9 gene editing-are discussed. Advancements in these novel therapies highlight a shift towards disease-modifying interventions. However, continued clinical and translational research is essential to develop a definitive cure.
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Efficient and crucial quality control of HAP1 cell ploidy status.
The near-haploid human cell line HAP1 recently became a popular subject for CRISPR/Cas9 editing, since only one allele requires modification. Through the gene-editing service at Horizon Discovery, there are at present more than 7500 edited cell lines available and the number continuously increases. The haploid nature of HAP1 is unstable as cultures become diploid with time. Here, we demonstrated some fundamental differences between haploid and diploid HAP1 cells, hence underlining the need for taking control over ploidy status in HAP1 cultures prior to phenotyping. Consequently, we optimized a procedure to determine the ploidy of HAP1 by flow cytometry in order to obtain diploid cultures and avoid ploidy status as an interfering variable in experiments. Furthermore, in order to facilitate this quality control, we validated a size-based cell sorting procedure to obtain the diploid culture more rapidly. Hence, we provide here two streamlined protocols for quality controlling the ploidy of HAP1 cells and document their validity and necessity.This article has an associated First Person interview with the co-first authors of the paper.
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