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Lipid Nanoparticles for In Vivo Lung Delivery of CRISPR-Cas9 Ribonucleoproteins Allow Gene Editing of Clinical Targets.
In the past 10 years, CRISPR-Cas9 has revolutionized the gene-editing field due to its modularity, simplicity, and efficacy. It has been applied for the creation of in vivo models, to further understand human biology, and toward the curing of genetic diseases. However, there remain significant delivery barriers for CRISPR-Cas9 application in the clinic, especially for in vivo and extrahepatic applications. In this work, high-throughput molecular barcoding techniques were used alongside traditional screening methodologies to simultaneously evaluate LNP formulations encapsulating ribonucleoproteins (RNPs) for in vitro gene-editing efficiency and in vivo biodistribution. This resulted in the identification of a lung-tropic LNP formulation, which shows efficient gene editing in endothelial and epithelial cells within the lung, targeting both model reporter and clinically relevant genomic targets. Further, this LNP shows no off-target indel formation in the liver, making it a highly specific extrahepatic delivery system for lung-editing applications.
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Simulation of CRISPR/Cas9-mediated gene editing for the Vitellogenin gene in Apis mellifera.
CRISPR/Cas9 genome editing provides a powerful framework for interrogating gene function in Apis mellifera. Yet, empirical application remains challenging due to biological constraints, including haplodiploid genetics, narrow embryonic injection window, and the social rearing requirements that complicate functional validation. These constraints necessitate in silico pre-screening to maximize editing success before resource-intensive wet-lab implementation. Within the omnigenic framework, which distinguishes core regulatory genes from peripheral loci buffered by network effects, vitellogenin (Vg) represents an optimal target which is ancestrally dedicated to yolk provisioning; it has been co-opted to orchestrate diverse non-reproductive functions including longevity, stress resistance, immunity, and social behavior. We developed a computational pipeline to design a list of 57 and 56 candidate guide RNAs (gRNA) for targeted Vg knockout, evaluating candidate sites in both functional exons 2 and 3 based on structural accessibility and frameshift efficiency. Comparative analysis revealed complementary strengths in two top-best candidates from initial target pool of predicted gRNAs. The gRNA targeting exon 2 exhibits weaker secondary structure (ΔG = -0.25 kcal/mol versus -2.10 kcal/mol for exon 3), aligning with empirical evidence that sites with ΔG > -1.0 kcal/mol achieve 2-5 × higher Cas9 binding efficiency. This site yielded moderate frameshift frequency (77.8%; 61.9 percentile). Conversely, the predicted editing outcome for the gRNA targeting exon 3, despite stronger structural constraints, demonstrated superior functional disruption metrics demonstrating very high frameshift frequency (88.3%; 95.2 percentile), high in silico editing precision, minimal microhomology-mediated repair bias, and reproducible outcomes wherein nearly all predicted indels disrupt the coding sequence. Protein structure and domain analyses further predict that frameshift edits will generate a truncated protein missing all downstream functional domains. We recommend parallel empirical validation of both exon 2 and exon 3 targets to resolve the trade-off between structural accessibility (favoring higher editing rates) and frameshift efficacy (favoring complete loss-of-function). This dual-target strategy accommodates uncertainty in in vivo performance while maximizing the probability of generating informative phenotypes. Our in silico framework enables rational CRISPR design in non-model organisms by computationally balancing biophysical accessibility with functional impact, accelerating functional genomics in species where empirical optimization faces substantial biological constraints.
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Bacterial nanotechnology.
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Research Progress on the Mechanism and Application of the Type I CRISPR-Cas System.
The CRISPR-Cas system functions as an adaptive immune mechanism in archaea and bacteria, providing defense against the invasion of foreign nucleic acids. Most CRISPR-Cas systems are classified into class 1 or class 2, with further subdivision into several subtypes. The primary distinction between class 1 and class 2 systems lies in the assembly of their effector modules. In class 1 systems, the effector complex consists of multiple proteins with distinct functions, whereas in class 2 systems, the effector is associated with a single protein. Class 1 systems account for approximately 90% of the CRISPR-Cas repertoire and are categorized into three types (type I, type IV, and type III) and 12 subtypes. To date, various CRISPR-Cas systems have been widely employed in the field of genetic engineering as essential tools and techniques for genome editing. Type I CRISPR-Cas systems remain a valuable resource for developing sophisticated application tools. This review provides a comprehensive review of the characteristics, mechanisms of action, and applications of class 1 type I CRISPR-Cas systems, as well as transposon-associated systems, offering effective approaches and insights for future research on the mechanisms of action, as well as the subsequent development and application of type I CRISPR-Cas systems.
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CRISPR-Cas9-based gene editing as a proof-of-concept approach in an inborn error of immunity caused by a DCLRE1C variant.
Hypomorphic DCLRE1C variants impair T and B cell development, leading to combined immunodeficiency (CID) or leaky severe combined immunodeficiency (SCID). Current treatment options, such as allogeneic hematopoietic stem cell transplantation (aHSCT), are associated with significant risks, highlighting the need for alternative therapeutic strategies. In this study, we report the first a proof-of-concept CRISPR-Cas9–mediated correction of a hypomorphic DCLRE1C variant (c.194 C > T; p.T65I) in CD4 + helper T (Th) cells using CRISPR-Cas9 gene-editing technology. CD4 + Th cells were isolated, and the variant region was edited with sgRNA and donor DNA. Gene editing efficiency was confirmed by Sanger sequencing, revealing successful restoration of the target region to its wild-type sequence. Functional analyses showed a significant increase in CD25 activation and Artemis protein expression post-editing, although DCLRE1C mRNA levels remained unchanged. The approximately 6–8% increase in CD25 expression was statistically significant but did not reach healthy control levels. These findings suggest that CRISPR-Cas9 –mediated gene editing may enable precise correction and induce measurable cellular-level functional changes, supporting biological feasibility rather than therapeutic efficacy. This study provides a foundation for future research on HSCs and underscores the potential role of CRISPR-Cas9–based approaches in the treatment of inborn errors of immunity (IEIs) associated with DCLRE1C variants.
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Pig Coat Color Manipulation by MC1R Gene Editing.
Black coat color in pigs is determined by the dominant E allele at the MC1R locus. Through comparing MC1R gene sequences between recessive e and dominant ED1 alleles, we identified four missense mutations that could affect MC1R protein function for eumelanin synthesis. With the aim of devising a genetic modification method for pig coat color manipulation, we mutated the e allele in the Duroc breed to the dominant ED1 allele using CRISPR-mediated homologous recombination for the four mutation substitutions at the MC1R locus. The MC1R-modified Duroc pigs generated using the allele replacement strategy displayed uniform black coat color across the body. A genotyping assay showed that the MC1R-modified Duroc pigs had a heterozygous ED1/e allele at the MC1R locus; in addition, the pigs remained in the Duroc genetic background. Our work offers a gene editing method for pig coat color manipulation, which could value the culture of new pig varieties meeting the needs of diversified market.
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CRISPR-Cas9 Applications in Cardiovascular Disease.
The CRISPR-Cas9 system is an economical and accessible gene-editing technology first discovered as a naturally occurring bacterial immune system. Since its fairly recent discovery, CRISPR-Cas9 system's efficiency and simplicity have been successfully used to edit genomes of living organisms in many fields, working in vitro and in vivo in germline and somatic cells to knock-out harmful mutated genes or in some cases working to knock-in a beneficial gene. A current application of the gene-editing system works against specific mutations that cause certain cardiovascular diseases. However, there are current technical limitations as well as ethical dilemmas in introducing gene-editing to humans. Here, we explore highlights on the current state of research of the CRISPR-Cas9 system through the lens of cardiovascular disease and examine potential untouched applications of the system in the field of cardiology.
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CRISPR/Cas9 genome editing for comparative genetic analysis related to soy sauce brewing in Aspergillus sojae industrial strains.
Aspergillus sojae has traditionally been used in soy sauce brewing. Genetic modification techniques have been established in A. sojae, but it is difficult to apply them to various industrial strains. Although we have previously developed a CRISPR/Cpf1 system for genetic modification of A. sojae, another genome editing system was required for versatile modification. In addition, repetitive genetic modification using the CRISPR system has not been established in A. sojae. In this study, we demonstrated mutagenesis, gene deletion/integration, and large deletion of a chromosomal region in A. sojae using the CRISPR/Cas9 system. We also successfully performed repetitive genetic modification using a method that involved forced recycling of genome-editing plasmids. Moreover, we demonstrated that the effects of genetic modification related to soy sauce brewing differed among A. sojae industrial strains. These results showed that our technique of using the CRISPR/Cas9 system is a powerful tool for genetic modification in A. sojae.
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Editing the Epigenome: Overview, Open Questions, and Directions of Future Development.
The introduction of CRISPR/Cas has resulted in a strong impulse for the field of gene-targeted epigenome reprogramming. In this approach EpiEditors are applied in cells, which consist of a DNA-binding part for targeting and a functional part to induce chromatin modifications at targeted genome loci. The accumulating evidence of epigenetic reprogramming of a given genomic locus resulting in gene expression changes indicated causal relationships of epigenetic marks instructing gene expression and opened the field for mainstream applications. In this perspective, an overview of the current status of the field is provided, including its applications and future perspectives. The dependence of critical parameters like specificity, effectivity, and sustainability of epigenome editing on experimental settings and conditions including the expression levels and the duration of the expression of the EpiEditors, their DNA-binding affinity and specificity, and the cross talk between EpiEditors and cellular chromatin modifiers is discussed. Once established in fully functional "plug-and-play" mode, epigenome editing will allow to better understand epigenetic expression control and to translate such knowledge into therapeutic tools.
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Photoactivatable nanoCRISPR/Cas9 System Based on crRNA Reversibly Immobilized on Carbon Nanoparticles.
Here, we proposed a new approach to engineering a photoactivatable CRISPR/Cas9 gene-editing system. The novel nanoCRISPR/Cas9 system is based on the use of auxiliary photocleavable oligodeoxyribonucleotides (PC-DNAs) complementary to crRNA. PC-DNAs contained up to three UV-sensitive linkers made of 1-(2-nitrophenyl)-1,2-ethanediol inside the oligonucleotide chain. Immobilizing PC-DNAs on the surface of carbon nanoparticles through 3'-terminal pyrene residue provided sufficient blocking of crRNA (and corresponding Cas9 activity) before UV irradiation and allows for crRNA release after UV irradiation at 365 nm, which restores Cas9 activity. We optimized the length of blocking photocleavable oligonucleotide, number of linkers, time of irradiation, and the type of carbon nanoparticles. Based on the results, we consider the nanoCRISPR/Cas9 system involving carbon-encapsulated iron nanoparticles the most promising. It provides the greatest difference of functional activity before/after irradiation and can be used in prospective for magnetic field-controlled delivery of CRISPR system into the target cells or tissues and spatiotemporal gene editing induced by UV irradiation.
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