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In recognition of the Spanish origin of CRISPR/Cas9. Implications for the treatment of familial heart disease. Response.
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CRISPRa-mediated transcriptional activation of the SlPR-1 gene in edited tomato plants.
With the continuous deterioration of arable land due to an ever-growing population, improvement of crops and crop protection have a fundamental role in maintaining and increasing crop productivity. Alternatives to the use of pesticides encompass the use of biological control agents, generation of new resistant crop cultivars, the application of plant activator agrochemicals to enhance plant defenses, and the use of gene editing techniques, like the CRISPR-Cas system. Here, we test the hypothesis that epigenome editing, via CRISPR activation (CRISPRa), activate tomato plant defense genes to confer resistance against pathogen attack. We provide evidence that edited tomato plants for the PATHOGENESIS-RELATED GENE 1 gene (SlPR-1) show enhanced disease resistance to Clavibacter michiganensis subsp. michiganensis infection. Resistance was assessed by evaluating disease progression and symptom appearance, pathogen accumulation, and changes in SlPR-1 gene expression at different time points. We determined that CRISPRa-edited plants develop enhanced disease-resistant to the pathogen without altering their agronomic characteristics and, above all, preventing the advancement of disease symptoms, stem canker, and plant death.
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CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates.
CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.
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Engineered Interactions with Mesoporous Silica Facilitate Intracellular Delivery of Proteins and Gene Editing.
Intracellular delivery of functional proteins is a promising, but challenging, strategy for many therapeutic applications. Here, we report a new methodology that overcomes drawbacks of traditional mesoporous silica (MSi) particles for protein delivery. We hypothesize that engineering enhancement in interactions between proteins and delivery vehicles can facilitate efficient encapsulation and intracellular delivery. In this strategy, surface lysines in proteins were modified with a self-immolative linker containing a terminal boronic acid for stimulus-induced reversibility in functionalization. The boronic acid moiety serves to efficiently interact with amine-functionalized MSi through dative and electrostatic interactions. We show that proteins of different sizes and isoelectric points can be quantitatively encapsulated into MSi, even at low protein concentrations. We also show that the proteins can be efficiently delivered into cells with retention of activity. Utility of this approach is further demonstrated with gene editing in cells, through the delivery of a CRISPR/Cas9 complex.
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Genome-engineering technologies for modeling and treatment of cystic fibrosis.
Cystic fibrosis (CF) is an autosomal recessive disease caused by defects in the CF transmembrane conductance regulator (CFTR) protein. Due to the genetic nature of the disease, interventions in the genome can target any underlying alterations and potentially provide permanent disease resolution. The current development of gene-editing tools, such as designer nuclease technology capable of genome correction, holds great promise for both CF and other genetic diseases. In recent years, Cas9-based technologies have enabled the generation of genetically defined human stem cell and disease models based on induced pluripotent stem cells (iPSC). In this article, we outline the potential and possibilities of using CRISPR/Cas9-based gene-editing technology in CF modeling.
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Advances in CRISPR-Cas systems for gut microbiome.
CRISPR-Cas technology has revolutionized microbiome research by enabling precise genetic manipulation of microbial communities. This review explores its diverse applications in gut microbiome studies, probiotic development, microbiome diagnostics, pathogen targeting, and microbial community engineering. Engineered bacteriophages and conjugative probiotics exemplify CRISPR-Cas's capability for targeted bacterial manipulation, offering promising strategies against antibiotic-resistant infections and other gut-related disorders. CRISPR-Cas systems also enhance probiotic efficacy by improving stress tolerance and colonization in the gastrointestinal tract. CRISPR-based techniques in diagnostics enable early intervention by enabling fast and sensitive pathogen identification. Furthermore, CRISPR-mediated gene editing allows tailored modification of microbial populations, mitigating risks associated with horizontal gene transfer and enhancing environmental and health outcomes. Despite its transformative potential, ethical and regulatory challenges loom large, demanding robust frameworks to guide its responsible application. This chapter highlights CRISPR-Cas's pivotal role in advancing microbiome research toward personalized medicine and microbial therapeutics while emphasizing the imperative of balanced ethical deliberations and comprehensive regulatory oversight.
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Efficient genome editing of two-cell mouse embryos via modified CRISPR/Cas electroporation.
Creating genetically modified (GM) animals using CRISPR/Cas mediated through the electroporation of two-cell stage embryos, rather than fertilized eggs, holds considerable potential. The full potential of genome editing using two-cell stage embryos is only beginning to be explored. We developed an improved electroporation method to prevent blastomere fusion in two-cell-stage embryos, enabling efficient genome editing. Using this method, we demonstrated that the indel mutation rates and ssODN knock-in (KI) efficiencies in two-cell-stage embryos are comparable to those in fertilized eggs, with a tendency for higher efficiency in long DNA KI. This study highlights the potential value of two-cell-stage embryos and provides enhanced animal model production opportunities. Furthermore, realizing genome editing in two-cell-stage embryos extends the editing timeframe from fertilized egg to two-cell-stage embryo, offering promising avenues for future research in embryo genome editing techniques.
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Genome Editing as a Treatment for the Most Prevalent Causative Genes of Autosomal Dominant Retinitis Pigmentosa.
: Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of diseases with more than 250 causative genes. The most common form is retinitis pigmentosa. IRDs lead to vision impairment for which there is no universal cure. Encouragingly, a first gene supplementation therapy has been approved for an autosomal recessive IRD. However, for autosomal dominant IRDs, gene supplementation therapy is not always pertinent because haploinsufficiency is not the only cause. Disease-causing mechanisms are often gain-of-function or dominant-negative, which usually require alternative therapeutic approaches. In such cases, genome-editing technology has raised hopes for treatment. Genome editing could be used to i) invalidate both alleles, followed by supplementation of the wild type gene, ii) specifically invalidate the mutant allele, with or without gene supplementation, or iii) to correct the mutant allele. We review here the most prevalent genes causing autosomal dominant retinitis pigmentosa and the most appropriate genome-editing strategy that could be used to target their different causative mutations.
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Gene Editing of Nicotiana benthamiana Architecture for Space-Efficient Production of Recombinant Proteins in Closed Environments.
Indoor vertical farming (VF) offers practical advantages for the cultivation of plant protein bio-factories including plant uniformity, product consistency, water/nutrient recycling and production cycles on a year-round basis. Much progress has been achieved toward the development of innovative systems for artificial lighting, automated irrigation, plant handling, environmental control and space use optimization in VF systems. Here, we used a CRISPR/Cas9 gene editing approach to generate mutant lines of protein expression host Nicotiana benthamiana presenting a compact, space-efficient phenotype suited to VF systems. Our strategy consisted of altering apical dominance by suppressing the synthesis of strigolactone, a negative regulator of axillary bud outgrowth. Strigolactone-depleted lines were generated by hindering the expression of either Carotenoid cleavage dioxygenase 7 (CCD7) or Carotenoid cleavage dioxygenase 8 (CCD8), two key enzymes of the strigolactone synthetic pathway. Knocking out of either enzyme had no impact on the plant's growth rate but drastically influenced its auxin/cytokinin ratio, leaf proteome and overall architecture. The ΔCCD mutants exhibited an axillary growth-oriented development pattern, altered glycolytic and malate-processing metabolic fluxes to drive pyruvate and cytokinin production, and a spatial footprint reduced by 45%-50% compared to the LAB strain commonly used for protein expression. Most importantly, recombinant protein yields per plant were maintained in the mutant lines, as illustrated with GFP and rituximab, a chimeric antibody of therapeutic value. Our data confirm the usefulness of ΔCCD7 and ΔCCD8 knockouts leading to strigolactone depletion for the generation of compact, space-efficient N. benthamiana lines better suited to VF systems.
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An overview of applications of CRISPR-Cas technologies in biomedical engineering.
Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one of the major genome editing systems and allows changing DNA levels of an organism. Among several CRISPR categories, the CRISPR-Cas9 system has shown a remarkable progression rate over its lifetime. Recently, other tools including CRISPR-Cas12 and CRISPR-Cas13 have been introduced. CRISPR-Cas9 system has played a key role in the industrial cell factory's production and improved our understanding of genome function. Additionally, this system has been used as one of the major genome editing systems for the diagnosis and treatment of several infectious and non-infectious diseases. In this review, we discuss CRISPR biology, its versatility, and its application in biomedical engineering.
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