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Optimization Strategies for the CRISPR-Cas9 Genome-Editing System.
The CRISPR-Cas9 system uncovered in bacteria has emerged as a powerful genome-editing technology in eukaryotic cells. It consists of two components-a single guide RNA (sgRNA) that directs the Cas9 endonuclease to a complementary DNA target site. Efficient targeting of individual genes requires highly active sgRNAs. Recent efforts have made significant progress in understanding the sequence features that increase sgRNA activity. In this introduction, we highlight advancements in the field of CRISPR-Cas9 targeting and discuss our web tool CRISPRscan, which predicts the targeting activity of sgRNAs and improves the efficiency of the CRISPR-Cas9 system for in vivo genome engineering.
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TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells.
CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.
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Plant Gene Knockout and Knockdown by CRISPR-Cpf1 (Cas12a) Systems.
CRISPR-Cpf1 (Cas12a) is a class II type V endonuclease, which has been used as a genome editing tool in different biological systems. Here we describe a fast, efficient, and user-friendly system for CRISPR-Cpf1 expression vector assembly. In this system, the Pol II promoter is used to drive the expression of both Cpf1 and its crRNA, with the crRNA flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozyme RNAs for precise crRNA processing. All the components of this system can be modified depending on plant species and experimental goals. Using this system, nearly 100% editing efficiency and 90% gene expression decrease were achieved in rice and Arabidopsis, respectively.
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Opto-CRISPR technologies in translational neuropharmacology.
Opto-CRISPR (optically controlled CRISPR/Cas) represents a major advancement in precision neurology by enabling localized gene editing within the nervous system at precise times and locations. While preclinical studies still rely largely on classical CRISPR/Cas systems, opto-CRISPR offers unprecedented opportunities to safely treat neurological diseases at their genetic roots. It achieves this by spatiotemporally correcting pathogenic mutations, halting toxic protein accumulation, repairing neurons, rebuilding neural circuitry, and accelerating drug discovery through highly realistic disease models. This review discusses this emerging field and its transformative potential for translational neuropharmacology, focusing on therapies for neurodevelopmental and neurodegenerative disorders. Opto-CRISPR is classified into four generations: Generation 1 (light-controlled Cas protein systems), Generation 2 (light-activated guide RNA systems), Generation 3 (light-controlled inhibitor systems), and Generation 4 (NIR upconversion and epigenetic multiplexing). Their working mechanisms, applications, advantages, and limitations are comprehensively described. We also critically discuss how opto-CRISPR offers distinct advantages over traditional biologics and remote modalities (e.g., small-molecule drugs, monoclonal antibodies, antisense oligonucleotides, viral-mediated gene therapy, magnetogenetics, and sonogenetics) in neuropharmacology. Finally, we address current challenges and the therapeutic outlook for the clinical translation of opto-CRISPR.
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Back to gene therapy: bringing the EU regulation into the future.
The current European framework for Advanced Therapy Medicinal Products (ATMPs), which also covers gene therapies, was established in 2007 at the dawn of the biotechnology era in the pharmaceutical sector. However, its inherent limitations have been revealed by the new frontiers enabled by the scientific and technological advances of the past few decades, particularly in the areas of innovative gene therapeutics (e.g., CRISPR/Cas9-RNA complexes), manufacturing technologies, and delivery systems. Here, we contribute to the scientific discussion on how to reduce regulatory uncertainty in the qualification of medicinal products with a different mechanism of action (e.g., editing the host genome or regulating gene expression), and on rationalizing regulatory support and incentives for the most complex therapies in terms of mechanism of action and manufacturing process.
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Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses.
Some prevention strategies, including vaccines and antibiotic alternatives, have been developed to reduce enterotoxigenic Escherichia coli proliferation in animal production. In this study, a wild-type strain of BE311 with a virulent heat-stable enterotoxin gene identical to E. coli K99 was isolated for its high potential for gene expression ability. The whole genome of E. coli BE311 was sequenced for gene analyses and editing. Subsequently, the fluorescent gene mCherry was successfully knocked into the genome of E. coli BE311 by CRISPR/Cas9. The E. coli BE311−mCherry strain was precisely quantified through the fluorescence intensity and red colony counting. The inflammatory factors in different intestinal tissues all increased significantly after an E. coli BE311−mCherry challenge in Sprague−Dawley rats (p < 0.05). The heat-stable enterotoxin gene of E. coli BE311 was knocked out, and an attenuated vaccine host E. coli BE311-STKO was constructed. Flow cytometry showed apoptotic cell numbers were lower following a challenge of IPEC-J2 cells with E. coli BE311-STKO than with E. coli BE311. Therefore, the E. coli BE311−mCherry and E. coli BE311-STKO strains that were successfully constructed based on the gene knock-in and knock-out technology could be used as ideal candidates in ETEC challenge models and for the development of attenuated vaccines.
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[Strategies for Optimizing Recombinant Protein Synthesis in Plant Cells: Classical Approaches and New Directions].
At present, pharmacologically significant proteins are synthesized in different expression systems, from bacterial to mammalian and insect cell cultures. The plant expression systems (especially suspension cell culture) combine the simplicity and low cost of bacterial systems with the ability to perform eukaryotic-type posttranslational protein modifications. A low (compared with bacterial systems) yield of the target recombinant protein is one of the shortcomings of the plant expression systems. In this review, methods, developed over the past two decades, to increase the level of recombinant gene expression and methods to prevent silencing, caused by a random insertion of the target gene into heterochromatin region, are considered. The emergence of CRISPR/Cas technologies led to the creation of a new approach to increase the gene expression level, directional insertion of "pharmaceutical" protein genes in specific, knowingly transcriptionally active genome regions. The plant cell housekeeping gene loci, actively expressed throughout the interphase, are these regions. The organization of some housekeeping genes, most promising for transferring recombinant protein genes in their loci, is considered in detail.
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Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates the ZNF334 gene to inhibit the growth of colorectal cancer.
Although therapeutic targets for colorectal cancer (CRC) treatment have been developed, the treatment outcomes are not ideal and survival rates for CRC patients remain low. It is critical to identify a specific target and develop an effective CRC treatment system. The ZNF334 gene is a newly identified member of Zinc-finger proteins (ZNFs), which is essential for key biological processes associated with tumorigenesis. Abnormal epigenetic reprogramming of the ZNF334 gene promoter region decreases its expression in CRC and further induces the occurrence of CRC. Here, we clarified that P300 in CRC can regulate the H3K9/27 ac in the ZNF334 promoter. Furthermore, histone acetylation of the ZNF334 promoter region was increased by dCas9-P300 to normalize the deficiency of ZNF334 expression, thereby inhibiting the growth of CRC. Collectively, our findings enable a facile way to affect gene expression using CRISPR/Cas9-based epigenome editing and further determine the causal link between histone acetylation and gene activation, providing a promising gene therapy strategy for the CRC treatment.
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Advancements in CRISPR-Cas Systems for Genome Editing towards Eradication of Human Microbial Pathogens.
CRISPR-Cas systems have been explored for targeted genome editing of several organisms. It is rapid, cost-effective, specific, and versatile technology. It requires expression of multidomain single Cas9 protein and single guide RNA (sgRNA) that targets desired nucleic acids in the presence of a protospacer adjacent motif (PAM). This generates a double-stranded break that is repaired by either non-homologous end joining or a homology-directed repair pathway. Currently, several Cas protein variants have been discovered and being used for several biotechnological applications. This review highlights the recent progress of CRISPR-Cas systems for genome editing of mainly human pathogenic microorganisms for their controlling infections.
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Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion.
Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells. Because of ultra-high efficiency of the CRISPR/Cas9 system with dual reporters and large DNA fragment deletion resulting from independent loci cleavage, monoclonal mutant cells could be easily identified by conventional PCR. In the speed genome editing method presented here, sophisticated genotyping methods are not necessary to identify loss of function mutations after CRISPR/Cas9 genome editing, and desirable loss of function mutant clones could be obtained in less than one month following transfection.
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