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Mutation Specific Treatments for Inherited Retinal Diseases.
The next frontier in genetic therapy for IRDs is the correction or silencing of specific mutations. This is of relevance to conditions with dominant negative and gain-of-function disease mechanisms, or with causative genes that are too large for gene replacement using AAV vectors. We discuss two approaches that have reached the stage of human clinical trials: CRISPR-Cas9 based gene editing and post-transcriptional gene silencing using antisense oligonucleotides. Other mutation-specific treatment approaches in pre-clinical development include adenosine deaminases acting on RNA (ADAR)-based RNA editing, RNA interference, and translational read through inducing drugs (TRADs). These have been reviewed elsewhere (e.g., Martinez Velazquez and Ballios 2021).
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CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency.
The CRISPR/Cas9 system is a powerful, revolutionary tool for genome editing. However, it is not without limitations. There are PAM-free and CRISPR-tolerant regions that cannot be modified by the standard CRISPR/Cas9 system, and off-target activity impedes its broader applications. To avoid these drawbacks, we developed a very simple CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique which does not require the construction of a plasmid to express a specific gRNA. Instead, a universal N20 sequence with a very high targeting efficiency is inserted into the E. coli chromosome by homologous recombination, which in turn undergoes a double-stranded break by CRISPR/Cas9 and induces an intra-chromosomal recombination event to accomplish the editing process. This technique was shown to be able to edit PAM-free and CRISPR-tolerant regions with no off-target effects in Escherichia coli. When applied to multi-locus editing, CAGO was able to modify one locus in two days with a near 100% editing efficiency. Furthermore, modified CAGO was used to edit large regions of up to 100 kbp with at least 75% efficiency. Finally, genome editing by CAGO only requires a transformation procedure and the construction of a linear donor DNA cassette, which was further simplified by applying a modular design strategy. Although the technique was established in E. coli, it should be applicable to other organisms with only minor modifications.
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CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass.
The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of d-xylose and l-arabinose from wheat bran, and d-galacturonic acid from sugar beet pulp.
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Simulation-Based Engineering of Time-Delayed Safety Switches for Safer Gene Therapies.
CRISPR-based gene editing is a powerful tool with great potential for applications in the treatment of many inherited and acquired diseases. The longer that CRISPR gene therapy is maintained within a patient, however, the higher the likelihood that it will result in problematic side effects such as off-target editing or immune response. One approach to mitigating these issues is to link the operation of the therapeutic system to a safety switch that autonomously disables its operation and removes the delivered therapeutics after some amount of time. We present here a simulation-based analysis of the potential for regulating the time delay of such a safety switch using one or two transcriptional regulators and/or recombinases. Combinatorial circuit generation identifies 30 potential architectures for such circuits, which we evaluate in simulation with respect to tunability, sensitivity to parameter values, and sensitivity to cell-to-cell variation. This modeling predicts one of these circuit architectures to have the desired dynamics and robustness, which can be further tested and applied in the context of CRISPR therapeutics.
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Research Progress on Nanoparticles-Based CRISPR/Cas9 System for Targeted Therapy of Tumors.
Cancer is a genetic mutation disease that seriously endangers the health and life of all human beings. As one of the most amazing academic achievements in the past decade, CRISPR/Cas9 technology has been sought after by many researchers due to its powerful gene editing capability. CRISPR/Cas9 technology shows great potential in oncology, and has become one of the most promising technologies for cancer genome-editing therapeutics. However, its efficiency and the safety issues of in vivo gene editing severely limit its widespread application. Therefore, developing a suitable delivery method for the CRISPR/Cas9 system is an urgent problem to be solved at present. Rapid advances in nanomedicine suggest nanoparticles could be a viable option. In this review, we summarize the latest research on the potential use of nanoparticle-based CRISPR/Cas9 systems in cancer therapeutics, in order to further their clinical application. We hope that this review will provide a novel insight into the CRISPR/Cas9 system and offer guidance for nanocarrier designs that will enable its use in cancer clinical applications.
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Focused ultrasound and microbubble-mediated delivery of CRISPR-Cas9 ribonucleoprotein to human induced pluripotent stem cells.
CRISPR-Cas9 ribonucleoproteins (RNPs) have been heavily considered for gene therapy due to their high on-target efficiency, rapid activity, and lack of insertional mutagenesis relative to other CRISPR-Cas9 delivery formats. Genetic diseases such as hypertrophic cardiomyopathy currently lack effective treatment strategies and are prime targets for CRISPR-Cas9 gene editing technology. However, current in vivo delivery strategies for Cas9 pose risks of unwanted immunogenic responses. This proof-of-concept study aimed to demonstrate that focused ultrasound (FUS) in combination with microbubbles can be used to deliver Cas9-sgRNA (single-guide RNA) RNPs and functionally edit human induced pluripotent stem cells (hiPSCs) in vitro, a model system that can be expanded to cardiovascular research via hiPSC-derived cardiomyocytes. Here, we first determine acoustic conditions suitable for the viable delivery of large proteins to hiPSCs with clinical Definity microbubble agents using our customized experimental platform. From here, we delivered Cas9-sgRNA RNP complexes targeting the EGFP (enhanced green fluorescent protein) gene to EGFP-expressing hiPSCs for EGFP knockout. Simultaneous acoustic cavitation detection during treatment confirmed a strong correlation between microbubble disruption and viable FUS-mediated protein delivery in hiPSCs. This study shows for the first time the potential for an FUS-mediated technique for targeted and precise CRISPR-Cas9 gene editing in human stem cells.
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Application of CRISPR/Cas9 system in breeding of new antiviral plant germplasm.
With the development and improvement of CRISPR/Cas9 system in genomic editing technology, the system has been applied to the prevention and control of animal viral infectious diseases, which has made considerable achievements. It has also been applied to the study of highly efficient gene targeting editing in plant virus genomes. The CRISPR/Cas9-mediated targeted gene modification has not only achieved the genome editing of plant DNA virus, but also showed the genome editing potential of plant RNA virus. In addition, the CRISPR/Cas9 system functions at the gene transcriptional and post-transcriptional level, indicating that the system could regulate the replication of plant viruses through different ways. Compared with other plant viral disease control strategies, this system is more accurate in genome editing, more stable in gene expression regulation, and has broader spectrum of resistance to virus disease. In this review, we summarized the advantages, main problems and development tendency of CRISPR/cas9 system in breeding of new antiviral plant germplasms.
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Gene editing of clock components in Solanum lycopersicum: Effects on gene expression, development, and productivity.
The circadian clock plays a crucial role in regulating key biological processes, including growth and development. While studies in the model plant Arabidopsis thaliana have significantly advanced our understanding of circadian function, recent research has also focused on crop species for improved yield and quality. In this study, we examined the rhythmic behavior and regulatory function of circadian clock components in tomato (Solanum lycopersicum). Time course analyses of gene expression over the circadian cycle revealed robust rhythmic oscillations in tomato leaves under free-running conditions. Comparative analyses showed similar peak phases for several clock genes in Arabidopsis and tomato, suggesting functional conservation. Rhythms in tomato fruits, however, showed reduced amplitude, slight phase changes, or arrhythmia, indicating organ-specific circadian variations. By using CRISPR-Cas9 gene editing strategies (clockcrispr), we also showed that proper clock gene expression is essential for setting the phase in tomato plants. Leaf movement analyses also showed a phase change in the clockcrispr lines, correlating with shorter or longer periods. The clockcrispr lines also displayed distinct growth and developmental phenotypes that differ from those reported in the Arabidopsis clock mutant counterparts. Our transcriptomic analyses identified species-specific regulation of key target genes. The results offer mechanistic insights into the conserved and divergent molecular pathways governing circadian phenotypic variations between Arabidopsis and tomato plants.
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Advances in application of CRISPR-Cas13a system.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins serve as an adaptive immune system that safeguards prokaryotes and some of the viruses that infect prokaryotes from foreign nucleic acids (such as viruses and plasmids). The genomes of the majority of archaea and about half of all bacteria contain various CRISPR-Cas systems. CRISPR-Cas systems depend on CRISPR RNAs (crRNAs). They act as a navigation system to specifically cut and destroy foreign nucleic acids by recognizing invading foreign nucleic acids and binding Cas proteins. In this review, we provide a brief overview of the evolution and classification of the CRISPR-Cas system, focusing on the functions and applications of the CRISPR-Cas13a system. We describe the CRISPR-Cas13a system and discuss its RNA-directed ribonuclease function. Meanwhile, we briefly introduce the mechanism of action of the CRISPR-Cas13a system and summarize the applications of the CRISPR-Cas13a system in pathogen detection, eukaryotes, agriculture, biosensors, and human gene therapy. We are right understanding of CRISPR-Cas13a has been broadened, and the CRISPR-Cas13a system will be useful for developing new RNA targeting tools. Therefore, understanding the basic details of the structure, function, and biological characterization of CRISPR-Cas13a effector proteins is critical for optimizing RNA targeting tools.
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[Will the CRISPR / CAS9 system change the genome of humanity?].
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