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Viral Vectors, Exosomes, and Vexosomes: Potential armamentarium for delivering CRISPR/Cas to cancer cells.
The underlying cause of cancer is genetic disruption, so gene editing technologies, particularly CRISPR/Cas systems can be used to go against cancer. The field of gene therapy has undergone many transitions over its 40-year history. Despite its many successes, it has also suffered many failures in the battle against malignancies, causing really adverse effects instead of therapeutic outcomes. At the tip of this double-edged sword are viral and non-viral-based vectors, which have profoundly transformed the way scientists and clinicians develop therapeutic platforms. Viruses such as lentivirus, adenovirus, and adeno-associated viruses are the most common viral vectors used for delivering the CRISPR/Cas system into human cells. In addition, among non-viral vectors, exosomes, especially tumor-derived exosomes (TDEs), have proven to be quite effective at delivering this gene editing tool. The combined use of viral vectors and exosomes, called vexosomes, seems to be a solution to overcoming the obstacles of both delivery systems.
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Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells.
The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.
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Co-CRISPR: A valuable toolkit for mutation enrichment in the gene editing of Spodoptera frugiperda.
The CRISPR/Cas9 system has been successfully applied in dozens of diverse species; although the screening of successful CRISPR/Cas9 editing events remains particularly laborious, especially for those that occur at relatively low frequency. Recently, a co-CRISPR strategy was proved to enrich the desired CRISPR events. Here, the co-CRISPR strategy was developed in the Fall armyworm, Spodoptera frugiperda, with kynurenine 3-monooxygenase gene (kmo) as a marker. The kmo mosaics induced by single-guide RNAs (sgRNAs)/Cas9 displayed the darker green color phenotype in larvae, compared with wild type (brown), and mosaic-eye adults were significantly acquired from the mosaic larvae group. In the kmo knockout strain, no significant difference was observed in larval development and adult reproduction. Acetylcholinesterase 2 (ace2) and Wnt1 were selected as target genes to construct the co-CRISPR strategy using kmo marker. By co-injection of kmo and ace2 sgRNAs, the mutant efficiency of ace2 was significantly increased in the kmo mosaic (larvae or adults) groups. Similarly, more malformed pupae with Wnt1 mutations were observed in the darker green larvae group. Taken together, these results demonstrated that kmo was a suitable visible marker gene for the application and extension of co-CRISPR strategy in Fall armyworm. Using darker green color in larvae or mosaic-eye in adults from kmo knockout as a marker, the mutant efficiency of a target gene could be enriched in a Fall armyworm group consisting of marked individuals. The co-CRISPR strategy is helpful for gene function studies by the knockout technique with no or lethal phenotypes.
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Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae.
The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites.
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Genome Editing of Corynebacterium glutamicum Using CRISPR-Cpf1 System.
Corynebacterium glutamicum, as an important microbial chassis, has great potential in industrial application. However, complicated genetic modification is severely slowed by lack of efficient genome editing tools. The Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been verified as a very powerful tool for mediating genome alteration in many microorganisms but cannot work well in C. glutamicum. We recently developed two Francisella novicida (Fn) CRISPR-Cpf1 assisted systems for genome editing via homologous recombination in C. glutamicum. Here, we describe the protocols and demonstrated that N iterative rounds of genome editing can be achieved in 3 N + 4 or 3 N + 2 days, respectively.
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Cas9 Ribonucleoproteins (RNPs) for Gene Deletion.
Genetic manipulations in pathogenic microorganisms, like Aspergillus fumigatus, allow us to study the function of genes in pathogenicity or other important traits. Classic genetic engineering tools in filamentous fungi rely on DNA fragment sub-cloning or fusion PCR to build a gene deletion cassette containing extended flanking regions of homology to promote efficient recombination. However, the introduction of CRISPR technology into fungal genetics has greatly simplified and accelerated the genome editing process. Here, we describe a simple and universal, one-step CRISPR-Cas9-mediated genetic tool employing repair templates containing microhomology regions. This gene targeting system displays high homologous recombination efficiency rates and can be easily implemented across different genetic backgrounds.
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CRISPR Gene Editing for Nucleotide Repeat Expansion Disorders: A Systematic Review of Preclinical and Clinical Evidence.
BACKGROUND: Incurable hereditary diseases such as Duchenne muscular dystrophy (DMD), Huntington's disease (HD), and myotonic dystrophy type 1 (DM1) fall into the nucleotide repeat expansion disorder (NRED) category. The discovery of CRISPR-Cas genome editing has paved the way toward hopeful strategies for accurate DNA-level repair. This systematic review presents preclinical data on the efficacy, molecular effects, and limitations of CRISPR-based treatments for NREDs. METHODS: As per Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines, systematic PubMed, Scopus, and Embase searches up to June 2025 identified studies that evaluated CRISPR-Cas systems in human-derived in vitro models of NREDs. Methodological Index for Non-Randomized Studies tool was used to score eligible studies by methodological quality. CRISPR platforms, delivery systems, gene targets, molecular endpoints, and functional rescue data were extracted and synthesized descriptively. RESULTS: Twenty-four out of 6510 records screened were included. They employed most of them to target specific DMD (n = 9), HD (n = 6), and DM1 (n = 3) with patient-derived induced pluripotent stem cells or differentiated myogenic/neuronal cells. Streptococcus pyogenes CRISPR-associated protein 9 as a nuclease was the most frequently used, although engineered Cas9 enzymes and dCas9 fusion proteins were also utilized to control transcription. Delivery was achieved through viral vectors (adeno-associated virus, lentivirus) and nonviral routes (plasmid, lipofection, electroporation). Uniform genomic editing, transcript rescue, and protein restoration were seen in CRISPR-mediated editing studies, and functional restoration was demonstrated for splicing correction and dystrophin restoration. Methodological flaws such as the absence of blinding, failure to follow up, and lack of full reporting of off-target effects limited robustness. CONCLUSION: CRISPR-Cas systems exhibit reproducible molecular and functional correction in NRED models with their translational potential. Methodological strength, whole safety profiling, and in vivo verification remain a necessity, however, before clinical translation.
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Interactive database of genome editing applications in crops and future policy making in the European Union.
European R&D in plant breeding is lagging behind, bound by strict genetically modified organism (GMO) regulations, applied to all crop varieties obtained with genome editing techniques. We developed an online database of worldwide genome editing applications in crops to support conclusions and to facilitate science-based policy making for this plant breeding innovation.
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PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells.
Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%-30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.
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Genetic Engineering of Human Keratinocytes Using CRISPR/Cas9 Ribonucleoprotein Complexes or Modified Cas9-Encoding mRNAs.
CRISPR/Cas9 is a straightforward genome-editing technique that is implemented across disciplines and research areas. However, in keratinocytes, CRISPR/Cas9 can be particularly difficult due to variable genome-editing efficiency, reduced cell viability, and difficulties during (sub)cloning of gene-edited keratinocyte populations. Here, we provide a step-by-step detailed protocol for the genetic manipulation of human (primary) keratinocytes, including widely accepted procedures for the analysis of CRISPR/Cas9 efficiency, (sub)cloning procedures to select heterozygous or homozygous keratinocytes, and off-target genome-editing analysis.
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