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CRISPR/Cas9-Mediated Genome Editing for Pseudomonas fulva, a Novel Pseudomonas Species with Clinical, Animal, and Plant-Associated Isolates.
As one of the most widespread groups of Gram-negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant-associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time-consuming and laborious endeavor. Here, by using the CRISPR-Cas system, a versatile gene-editing tool with a two-plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic-related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked-in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species.
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Evolutionary Biology and Gene Editing of Cat Allergen, Fel d 1.
Allergy to domestic cat affects up to 15% of the population, and sensitization to cat allergen is associated with asthma. Despite the pervasiveness of cat allergic disease, current treatments have limited impact. Here, we present a bioinformatics analysis of the major cat allergen, Fel d 1, and demonstrate proof of principle for CRISPR gene editing of the allergen. Sequence and structural analyses of Fel d 1 from 50 domestic cats identified conserved coding regions in genes CH1 and CH2 suitable for CRISPR editing. Comparative analyses of Fel d 1 and orthologous sequences from eight exotic felid species determined relatively low-sequence identities for CH1 and CH2, and implied that the allergen may be nonessential for cats, given the apparent lack of evolutionary conservation. In vitro knockouts of domestic cat Fel d 1 using CRISPR-Cas9 yielded editing efficiencies of up to 55% and found no evidence of editing at predicted potential off-target sites. Taken together, our data indicate that Fel d 1 is both a rational and viable candidate for gene deletion, which may profoundly benefit cat allergy sufferers by removing the major allergen at the source.
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Recent advances in globin research using genome-wide association studies and gene editing.
A long-sought goal in the hemoglobin field has been an improved understanding of the mechanisms that regulate the switch from fetal (HbF) to adult (HbA) hemoglobin during development. With such knowledge, the hope is that strategies for directed reactivation of HbF in adults could be devised as an approach to therapy for the β-hemoglobinopathies thalassemia and sickle cell disease. Recent genome-wide association studies (GWAS) led to identification of three loci (BCL11A, HBS1L-MYB, and the β-globin cluster itself) in which natural genetic variation is correlated with different HbF levels in populations. Here, the central role of BCL11A in control of HbF is reviewed from the perspective of how findings may be translated to gene therapy in the not-too-distant future. This summary traces the evolution of recent studies from the initial recognition of BCL11A through GWAS to identification of critical sequences in an enhancer required for its erythroid-specific expression, thereby highlighting an Achilles heel for genome editing.
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Challenges for gene editing in common variable immunodeficiency disorders: Current and future prospects.
The original CRISPR Cas9 gene editing system and subsequent innovations offers unprecedented opportunities to correct severe genetic defects including those causing Primary Immunodeficiencies (PIDs). Common Variable Immunodeficiency Disorders (CVID) are the most frequent symptomatic PID in adults and children. Unlike many other PIDs, patients meeting CVID criteria do not have a definable genetic defect and cannot be considered to have an inborn error of immunity (IEI). Patients with a CVID phenotype carrying a causative mutation are deemed to have a CVID-like disorder consequent to an IEI. Patients from consanguineous families often have highly penetrant early-onset autosomal recessive forms of CVID-like disorders. Individuals from non-consanguineous families may have autosomal dominant CVID-like disorders with variable penetrance and expressivity. This essay explores the potential clinical utility as well as the current limitations and risks of gene editing including collateral genotoxicity. In the immediate future the main application of this technology is likely to be the in vitro investigation of epigenetic and polygenic mechanisms, which are likely to underlie many cases of CVID and CVID-like disorders. In the longer-term, the CRISPR Cas9 system and other gene-based therapies could be utilized to treat CVID-like disorders, where the underlying IEI is known.
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Engineered Cas9 exosome vesicles as a novel gene editing tool for targeted ASPN editing in osteoarthritis.
CRISPR-Cas9, an innovative genome-editing technique, holds immense promise in therapeutic applications; nevertheless, the lack of effective delivery methods for in vivo gene editing limits its utility in osteoarthritis (OA) treatment. Recently, exosomes, naturally derived nanosized vesicles secreted by cells, have attracted significant attention as potential vehicles for therapeutic cargo delivery. This study proposes a bioinspired engineered exosome-mediated CRISPR/Cas9 delivery platform for targeted editing of the Asporin (ASPN) gene as a potential precision therapy for OA. Specifically, chondrocyte affinity peptide (Cap)-modified MSC-derived exosomes were employed as natural, biocompatible carriers to deliver CRISPR/Cas9 components specifically to OA-affected chondrocytes, thereby achieving precise and efficient ASPN knockout. Flow cytometry analysis confirmed a modification efficiency of 79.1% for Cap, while the encapsulation efficiency of the ASPN-Cas9 plasmid into exosomes reached 9.5% ± 0.6%. Both in vivo and in vitro investigations revealed that this delivery approach markedly improved cellular uptake and gene-editing efficacy, achieving a substantial reduction of ASPN expression by 61.7%. This, in turn, alleviated ferroptosis, improved mitochondrial function, reduced chondrocyte senescence, inhibited inflammation, and enhanced the cartilage microenvironment. Altogether, these findings strongly suggest the promising therapeutic efficacy of this method in OA models, emphasizing its potential as a precise gene-targeting therapeutic intervention for OA.
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Conditional Expression of Cas9 and dCas9 in Lucilia cuprina Reveals dCas9-Associated Lethality.
Conditional sex transformation systems could improve genetic control strategies against insect pests. Here, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina. Using Tet-Off-regulated expression of Cas9 and dCas9, we targeted the sex-determining gene transformer with the goal of converting females into males. Conditional Cas9 expression enabled knockout of a visual marker gene, confirming inducible genome editing. However, strains expressing transformer-targeting sgRNA arrays did not undergo sex transformation. Embryonic microinjection of transformer-targeting sgRNAs into Cas9-expressing embryos produced intersex individuals, indicating that sgRNA expression from the integrated arrays was insufficient to disrupt the sex determination pathway. In contrast, high-level dCas9 expression was associated with developmental delays, reduced body weight, and lethality. These findings establish the first conditional CRISPR expression system in L. cuprina and demonstrate that Cas9 is compatible with inducible gene editing, whereas dCas9 is poorly tolerated at high expression levels.
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Therapeutic Germline Editing: Sense and Sensibility.
Safe and effective heritable editing of the human genome is years away from the clinic because of formidable technical, statutory, regulatory, and societal challenges. In particular, we note the fledgling state of the science, the imperatives of editing efficiency, specificity, and uniformity, and the extant legal roadblock.
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A homology independent sequence replacement strategy in human cells using a CRISPR nuclease.
Precision genomic alterations largely rely on homology directed repair (HDR), but targeting without homology using the non-homologous end-joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double-strand break in both dividing and non-dividing cells. Here, we demonstrate the use of NHEJ repair to replace genomic segments with donor sequences; we name this method 'Replace' editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9, we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci in cell lines and show successful exchange of exons in 16-54% of human cells. Using linear amplification methods and deep sequencing, we quantify the diversity of outcomes following Replace editing and profile the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.
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CRISPR/Cas9 in the era of nanomedicine and synthetic biology.
The CRISPR/Cas system was first discovered as a defense mechanism in bacteria and is now used as a tool for precise gene-editing applications. Rapidly evolving, it is increasingly applied in therapeutics. However, concerns about safety, specificity, and delivery still limit its potential. In this context, we introduce the concept of nanogenetics and speculate how the rational engineering of the CRISPR/Cas machinery could advance the biomedical field. In nanogenetics, the advantages of traditional approaches of synthetic biology could be expanded by nanotechnology approaches, enabling the design of a new generation of intrinsically safe and specific genome-editing platforms.
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Genome editing using artificial site-specific nucleases in zebrafish.
Zebrafish is a model vertebrate suitable for genetic analysis. Forward genetic analysis via chemical mutagenesis screening has established a variety of zebrafish mutants that are defective in various types of organogenesis, and the genes responsible for the individual mutants have been identified from genome mapping. On the other hand, reverse genetic analysis via targeted gene disruption using embryonic stem (ES) cells (e.g., knockout mouse) can uncover gene functions by investigating the phenotypic effects. However, this approach is mostly limited to mice among the vertebrate models because of the difficulty in establishing ES cells. Recently, new gene targeting technologies, such as the transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have been developed: that can directly introduce genome modifications at the targeted genomic locus. Here, we summarize these new and powerful genome editing techniques for the study of zebrafish.
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