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LION: a simple and rapid method to achieve CRISPR gene editing.
The RNA-guided CRISPR-Cas9 technology has paved the way for rapid and cost-effective gene editing. However, there is still a great need for effective methods for rapid generation and validation of CRISPR/Cas9 gRNAs. Previously, we have demonstrated that highly efficient generation of multiplexed CRISPR guide RNA (gRNA) expression array can be achieved with Golden Gate Assembly (GGA). Here, we present an optimized and rapid method for generation and validation in less than 1 day of CRISPR gene targeting vectors. The method (LION) is based on ligation of double-stranded gRNA oligos into CRISPR vectors with GGA followed by nucleic acid purification. Using a dual-fluorescent reporter vector (C-Check), T7E1 assay, TIDE assay and a traffic light reporter assay, we proved that the LION-based generation of CRISPR vectors are functionally active, and equivalent to CRISPR plasmids generated by traditional methods. We also tested the activity of LION CRISPR vectors in different human cell types. The LION method presented here advances the rapid functional validation and application of CRISPR system for gene editing and simplified the CRISPR gene-editing procedures.
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Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering.
Chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1, but has not been investigated extensively. In this report, we demonstrate that the gRNAs of Cas9 and Cpf1, and donor DNA can be chemically modified at their terminal positions without losing activity. Moreover, we show that 5' fluorescently labeled donor DNA can be used as a marker to enrich HDR edited cells by a factor of two through cell sorting. In addition, we demonstrate that the gRNA and donor DNA can be directly conjugated together into one molecule, and show that this gRNA-donor DNA conjugate is three times better at transfecting cells and inducing HDR, with cationic polymers, than unconjugated gRNA and donor DNA. The tolerance of the gRNA and donor DNA to chemical modifications has the potential to enable new strategies for genome engineering.
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[The CRISPR/Cas system: a genome editing tool to develop animal models of viral infections].
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Impact of Gene Editing Tools, Like CRISPR/Cas9, on the Public Health Response to Disease Outbreaks.
The purpose of this communication is to explore the implications of genome editing techniques, such as CRISPR/Cas9, on public health-related responses to outbreaks of disease. The recent commercialization of genome editing techniques makes the creation and release of genetically altered pathogens a much easier task, increasing the possibility to the point of needing discussion. Three areas need to be addressed: predictions concerning potential genetic alterations, predictions and implications concerning the release of genetically altered pathogens, and the short- and long-term implications of the release of genetically altered pathogens. Full discourse on these topics among professionals in the area of public health will help to combat harm from the use of any genetically altered biologic weapons. The topics covered here include a review of the CRISPR/Cas9 gene editing technique, including a discussion of which possibilities utilize genome editing. We then address predictions about the application of gene alterations in the context of bioweapons. We discuss a few basic concepts about the evolution of an intentionally released genetically altered organism based on circumstances and patterns gleaned from observing nature in the hope that this will aid in the public health response to bioterrorism attack. (Disaster Med Public Health Preparedness. 2017;11:155-159).
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Gasdermin E as a potential target and biomarker for CRISPR-Cas9-based cancer therapy.
Gasdermin E (GSDME), a protein pivotal in mediating pyroptosis, has gained significant attention due to its role in cancer pathogenesis and its potential as a therapeutic target. The advent of CRISPR-Cas9, a precise genome editing tool, has revolutionized cancer therapy by enabling the manipulation of GSDME expression and function. This review explores the interplay of GSDME and CRISPR-Cas9 in cancer, emphasizing GSDME's unique mechanism of cleavage-dependent pore formation in the cell membrane and its emerging applications as both a therapeutic target and a diagnostic biomarker. We discuss the potential and challenges of using GSDME-induced pyroptosis as a therapeutic strategy and how can enhance its efficacy and specificity. We conclude by highlighting promising future research directions in this emerging field.
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The SpRY Cas9 variant release the PAM sequence constraint for genome editing in the model plant Physcomitrium patens.
Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. Here we assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, we developed a SpRYCas9i variant, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. We show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and we could not detect off-target activity for the sgRNAs tested in this study. These findings contribute to advancing versatile genome editing techniques in plants.
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Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways.
Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.
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Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing.
Chimeric Antigen Receptor (CAR) T-cells represent a breakthrough in personalized cancer therapy. In this strategy, synthetic receptors comprised of antigen recognition, signaling, and costimulatory domains are used to reprogram T-cells to target tumor cells for destruction. Despite the success of this approach in refractory B-cell malignancies, optimal potency of CAR T-cell therapy for many other cancers, particularly solid tumors, has not been achieved. Factors such as T-cell exhaustion, lack of CAR T-cell persistence, cytokine-related toxicities, and bottlenecks in the manufacturing of autologous products have hampered the safety, effectiveness, and availability of this approach. With the ease and accessibility of CRISPR-Cas9-based gene editing, it is possible to address many of these limitations. Accordingly, current research efforts focus on precision engineering of CAR T-cells with conventional CRISPR-Cas9 systems or novel editors that can install desired genetic changes with or without introduction of a double-stranded break (DSB) into the genome. These tools and strategies can be directly applied to targeting negative regulators of T-cell function, directing therapeutic transgenes to specific genomic loci, and generating reproducibly safe and potent allogeneic universal CAR T-cell products for on-demand cancer immunotherapy. This review evaluates several of the ongoing and future directions of combining next-generation CRISPR-Cas9 gene editing with synthetic biology to optimize CAR T-cell therapy for future clinical trials toward the establishment of a new cancer treatment paradigm.
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Application of CRISPR/Cas Technology in Spermatogenesis Research and Male Infertility Treatment.
As the basis of animal reproductive activity, normal spermatogenesis directly determines the efficiency of livestock production. An in-depth understanding of spermatogenesis will greatly facilitate animal breeding efforts and male infertility treatment. With the continuous development and application of gene editing technologies, they have become valuable tools to study the mechanism of spermatogenesis. Gene editing technologies have provided us with a better understanding of the functions and potential mechanisms of action of factors that regulate spermatogenesis. This review summarizes the applications of gene editing technologies, especially CRISPR/Cas9, in deepening our understanding of the function of spermatogenesis-related genes and disease treatment. The problems of gene editing technologies in the field of spermatogenesis research are also discussed.
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Efficient CRISPR/Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters.
CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.
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