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Gene editing of the wheat homologs of TONNEAU1-recruiting motif encoding gene affects grain shape and weight in wheat.
Grain size and weight are important components of a suite of yield-related traits in crops. Here, we showed that the CRISPR-Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1-recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double-copy mutant showing larger effect than the respective single copy mutants. The TaGW7-centered gene co-expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co-localization of TaGW7 with α- and β-tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7-associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR-Cas9 system with cross-species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.
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Genome editing: A novel approach to manage insect vectors of plant viruses.
Insect vectors significantly threaten global agriculture by transmitting numerous plant viruses. Various measures, from conventional insecticides to genetic engineering, are used to mitigate this threat. However, none provide complete resistance. Therefore, researchers are looking for novel control options. In recent years with the advancements in genomic technologies, genomes and transcriptomes of various insect vectors have been generated. However, the lack of knowledge about gene functions hinders the development of novel strategies to restrict virus spread. RNA interference (RNAi) is widely used to elucidate gene functions, but its variable efficacy hampers its use in managing insect vectors and plant viruses. Genome editing has the potential to overcome these challenges and has been extensively used in various insect pest species. This review summarizes the progress and potential of genome editing in plant virus vectors and its application as a functional genomic tool to elucidate virus-vector interactions. We also discuss the major challenges associated with editing genes of interest in insect vectors.
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Non-nuclear localization signal-guided CRISPR/Cas9 ribonucleoproteins for translocation and gene editing via apoferritin delivery vectors.
Direct delivery of the Cas9/sgRNA ribonucleoprotein (RNP) via appropriate carriers has been proved to be an important advance for the in vivo translocation and gene editing of CRISPR/Cas9. These carriers often require the nuclear localization signal (NLS) to fuse with Cas9 or the NLS-bearing protein to form a complex with Cas9 to enter the nucleus. In this study, we introduced apoferritin nanocages as carriers and DOX as a nuclear trigger for the nuclear transport of the Cas9/sgRNA ribonucleoprotein without the NLS (RNP-). Our experiments showed that loading RNP- and DOX into 4L-FTH subunit-based apoferritin nanocages leads to efficient endocytosis and lysosomal escape. Specifically, when DOX was administered at a concentration of 1 μM, we observed the activation of cellular defense mechanisms, which effectively facilitated the translocation of 4L-HFn@RNP-/DOX nanoparticles into the nucleus, thereby enabling intranuclear RNP- delivery. This strategy has been empirically demonstrated to achieve gene editing efficiencies of approximately 33% for the Lcn2 gene in MDA-MB-231 cells and 17.9% for the copepod green fluorescent protein (copGFP) gene in HeLa.copGFP cells in vitro. Moreover, in vivo editing efficacy, as tested in a HeLa.copGFP nude mouse model, was confirmed to be 16%. This delivery system presents a novel therapeutic approach for the nuclear delivery of small molecules or nucleic acid drugs, potentially overcoming the challenges associated with nuclear entry barriers.
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Moving towards a cure in genetics: what is needed to bring somatic gene therapy to the clinic?
Clinical trials using somatic gene editing (e.g., CRISPR-Cas9) have started in Europe and the United States and may provide safe and effective treatment and cure, not only for cancers but also for some monogenic conditions. In a workshop at the 2018 European Human Genetics Conference, the challenges of bringing somatic gene editing therapies to the clinic were discussed. The regulatory process needs to be considered early in the clinical development pathway to produce the data necessary to support the approval by the European Medicines Agency. The roles and responsibilities for geneticists may include counselling to explain the treatment possibilities and safety interpretation.
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CRISPR/Cas in gynecologic cancers: A review of experimental and therapeutic applications.
Gynecological malignancies-including cervical, ovarian, and endometrial cancers-remain a major global health challenge, contributing significantly to cancer-related morbidity and mortality among women. Despite advances in conventional treatments such as surgery, chemotherapy, radiotherapy, and immunotherapy, issues such as drug resistance, tumor recurrence, and limited efficacy in advanced-stage disease necessitate novel therapeutic strategies. The emergence of CRISPR/Cas-based genome editing has revolutionized cancer research by enabling precise, efficient, and programmable modifications of specific genomic loci. In gynecologic oncology, CRISPR/Cas systems have been employed to dissect oncogenic mechanisms, identify therapeutic targets, and develop innovative treatment modalities. In cervical cancer, CRISPR-mediated targeting of HPV E6 and E7 oncogenes has shown potential in restoring tumor suppressor pathways and enhancing chemosensitivity. In ovarian cancer, gene editing has been used to modulate chemoresistance, tumor angiogenesis, and metastasis through the knockout of key regulators such as DNMT1, EGFL6, and BRCA1/2. Similarly, in endometrial cancer, CRISPR tools have elucidated mechanisms of hormonal resistance and facilitated the development of in vivo models via somatic gene editing. This review highlights recent advances in the application of CRISPR/Cas technology to gynecologic malignancies, discussing its potential as both a therapeutic and research platform while acknowledging current limitations and translational hurdles.
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Broadening the targetable space: engineering and discovery of PAM-flexible Cas proteins.
The application of CRISPR-Cas systems has been hindered by their requirement for long protospacer-adjacent motifs (PAMs). Recent engineering and discovery of PAM-flexible Cas proteins have substantially broadened the targetable DNA sequence space, thereby facilitating genome editing and improving derivative technologies such as gene regulation, seamless cloning, and large-scale genetic screens.
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Gene editing and CRISPR-dependent homology-mediated end joining.
Gene editing is the intentional modification of a genetic locus in a living cell and is used for two general applications of great importance and wide interest. One is the inactivation of genes ('knockouts'), a process utilized to delineate the loss-of-function phenotype(s) of a particular gene. The second application ('knock-ins') is essentially the process of gene therapy, which predominately involves correcting a pre-existing mutated allele(s) of a gene back to wild-type to ameliorate some pathological phenotype associated with the mutation. Importantly, although these applications are conceptually exact reciprocal opposites of one another, they are achieved via mechanistically different pathways. In the case of knockouts, breakage (usually in the form of double-stranded breaks) of the chromosomal DNA at the site of targeting is used to engage a repair process (nonhomologous end joining) that is error prone. The ensuing repair frequently results in insertions/deletions at the cleavage site, which, in turn, results in out-of-frame mutations and, hence, a knockout of the gene in question. In the case of knock-ins, breakage (again, usually in the form of double-stranded breaks) of the DNA is used to engage a repair process (homology-dependent repair/recombination) in which homologous sequences between an incoming donor DNA (containing new genetic information) and the chromosomal DNA are exchanged. Although homology-directed repair was known to predominate in bacteria and lower eukaryotes, the competing process of nonhomologous end joining predominates in higher eukaryotes and was presumed to prevent the use of knock-in gene editing in human somatic cells in culture. A series of molecular and technical advances disproved this notion but still resulted in a process that was cumbersome, labor intensive, highly inefficient and slow. In 2013, however, a new RNA-programmable nuclease, CRISPR-Cas9 was described that has revolutionized the field and made gene editing accessible to anyone with even a rudimentary knowledge of molecular biology. Thus, gene editing in a wide variety of model organisms, as well as human somatic cells in culture, has become not only extremely feasible but also extremely facile, and it harbingers a golden age for directed mutagenesis, directed evolution and improvements in gene therapy.
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PEGylated Chitosan for Nonviral Aerosol and Mucosal Delivery of the CRISPR/Cas9 System in Vitro.
Chitosan has been widely employed to deliver nucleic acids such as siRNA and plasmids. However, chitosan-mediated delivery of a gene-editing system has not been reported yet. In this study, poly(ethylene glycol) monomethyl ether (mPEG) was conjugated to chitosan with different molecular weights (low molecular weight and medium molecular weight chitosan) achieving a high degree of substitution as identified by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR) spectra. PEGylated chitosan/pSpCas9-2A-GFP nanocomplexes were formed at different N/ P (amine group to phosphate group) ratios and characterized in terms of size and zeta potential. The nanocomplexes developed showed the capability to protect loaded nucleic acids from DNase I digestion and from the stresses of nebulization. In addition, we demonstrated that the PEG conjugation of chitosan improved the mucus-penetration capability of the formed nanocomplexes at N/ P ratios of 5, 10, 20, and 30. Finally, PEGylated low molecular weight chitosan nanocomplexes showed optimal transfection efficiency at an N/ P ratio of 20, while PEGylated medium molecular weight chitosan nanocomplexes showed an optimal transfection efficiency at an N/ P ratio of 5 at pH 6.5 and 6.8. This study established the basis for the delivery of a gene-editing system by PEGylated chitosan nanocomplexes.
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Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula.
An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula. CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and M. truncatula (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target phytoene desaturase (PDS) genes were designed. Agrobacterium-mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in M. truncatula. Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and mono-allelic homozygous mutants in M. truncatula. Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in PDS gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.
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Solid-phase reverse transfection for intracellular delivery of functionally active proteins.
Delivery of large and functionally active biomolecules across cell membranes presents a challenge in cell biological experimentation. For this purpose, we developed a novel solid-phase reverse transfection method that is suitable for the intracellular delivery of proteins into mammalian cells with preservation of their function. We show results for diverse application areas of the method, ranging from antibody-mediated inhibition of protein function to CRISPR/Cas9-based gene editing in living cells. Our method enables prefabrication of "ready to transfect" substrates carrying diverse proteins. This allows their easy distribution and standardization of biological assays across different laboratories.
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