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Harnessing CRISPR technology for viral therapeutics and vaccines: from preclinical studies to clinical applications.
The CRISPR/Cas system, identified as a type of bacterial adaptive immune system, have attracted significant attention due to its remarkable ability to precisely detect and eliminate foreign genetic material and nucleic acids. Expanding upon these inherent capabilities, recent investigations have unveiled the potential of reprogrammed CRISPR/Cas 9, 12, and 13 systems for treating viral infections associated with human diseases, specifically targeting DNA and RNA viruses, respectively. Of particular interest is the RNA virus responsible for the recent global outbreak of coronavirus disease 2019 (COVID-19), which presents a substantial public health risk, coupled with limited efficacy of current prophylactic and therapeutic techniques. In this regard, the utilization of CRISPR/Cas technology offers a promising gene editing approach to overcome the limitations of conventional methods in managing viral infections. This comprehensive review provides an overview of the latest CRISPR/Cas-based therapeutic and vaccine strategies employed to combat human viral infections. Additionally, we discuss significant challenges and offer insights into the future prospects of this cutting-edge gene editing technology.
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High-efficiency of genetic modification using CRISPR/Cpf1 system for engineered CAR-T cell therapy.
Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated promising efficacy in several kinds of blood cancers, including diffuse large B-cell lymphoma and acute and chronic lymphoblastic leukemia, etc. It is essential to effectively generate more potent and safer CAR-T cells through gene editing technologies for immune cell therapy. Conventional methods based on lentivirus, retrovirus and transposon, randomly integrate CAR sequence into T cell genome, which could lead to safety issues. Therefore, precise knock-in of CAR cassette into specific gene locus like TRAC and PDCD1 can lower the risks caused by random integration, as well as enhance the stability and function of the modified CAR-T cells. Current approaches of CRISPR/Cas9-based gene-editing have limitations in knock-in efficiency of the chimeric antigen receptor, while Cpf1, a CRISPR-Cas/RNA-guided nuclease, shows higher homology-directed repair (HDR) rate compared to Cas9 due to its unique biochemical characteristics. Here, we introduce a method combining electroporation and adeno-associated virus (AAV) infection to deliver CRISPR/Cpf1 components and a HDR template into T cells, thus precisely integrate CAR sequence at a specific gene locus with high efficiency.
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Peptide-assisted lipofection enables efficient non-viral delivery of large CRISPR/Cas9 constructs for genome editing applications.
Efficient and safe delivery of large genetic constructs such as CRISPR/Cas9 plasmids remains a critical bottleneck in gene therapy. In this work, the peptide-assisted lipofection (PAL) system was developed as a breakthrough non-viral vector for gene delivery, specifically designed for large plasmids including CRISPR/Cas9 constructs. This system achieved exceptional transfection efficiency up to 98.7 % in HEK293T cells, surpassing conventional delivery methods such as electroporation and standard lipofections. PAL successfully delivered large plasmids up to 29 kb while maintaining high cell viability and achieved 44.1 % indel formation efficiency in gene editing experiments. Fluorescence microscopy verified PAL's efficient endosomal escape and nuclear targeting abilities. The superior performance of the PAL is attributed to its cellular uptake and endosomal escape enhanced by transfection-assisting peptide. In vivo studies in mouse models showed sustained gene expression in liver tissue, demonstrating superior performance compared to naked plasmid delivery. These results establish PAL as a versatile and promising platform for gene therapy and genome editing, offering a safer alternative to viral vectors for large genetic payload delivery.
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CRISPR-Based Gene Therapy for Brain Disease.
Neurological disorders are complex and often very challenging for patients. Many of these conditions result from mutations in genes that are essential for normal function. Most existing treatments only alleviate symptoms, highlighting the urgent need for more effective therapeutic strategies. In the current drug development landscape, gene therapy offers hope as a promising approach. Specifically, CRISPR-Cas9 technology enables precise gene editing across diverse cell types and organisms. An increasing number of research groups are investigating innovative therapies and the molecular mechanisms behind neurological diseases. This review highlights the use of CRISPR-based gene therapies for various brain diseases, including multiple sclerosis, Alzheimer's, Parkinson's disease, epilepsy, stroke, and brain tumors. It consistently recognizes significant challenges in clinical applications, including overcoming the blood-brain barrier (BBB), managing off-target effects, ensuring efficient delivery, and addressing immunogenicity and ethical concerns.
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Targeted genome engineering based on CRISPR/Cas9 system to enhance FVIII expression in vitro.
BACKGROUND: Hemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease. METHODS: Initially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression. RESULTS: We identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro. CONCLUSIONS: Knock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases.
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Removing the toxic enantiomer from crop products with selective gene editing.
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Efficient genome editing using CRISPR/Cas9 technology and its application for identifying Sesquiterpene synthases involved in the biosynthesis of Steperoxides in Steccherinum ochraceum.
CRISPR technology has been widely used for gene editing in various species，but the genetic manipulation in basidiomycete mushrooms is still notoriously difficult for unknown endogenous promoters and inefficient DNA delivery. Steccherinum ochraceum is a white rot basidiomycete fungus with abundant secondary metabolites and plays an important ecological role worldwide. To facilitate the study of gene function in S. ochraceum, an effective CRISPR/Cas9 system was successfully developed by identifying highly efficient endogenous promoters, and utilizing the Agrobacterium-transformation method. Two efficient endogenous RNA polymerase II promoters (Psogpd and Psotef1) and one efficient RNA polymerase III promoter (Pu6-d) were identified and characterized, with an editing efficiency of 61.5 % at the ura3 locus. Using this optimized system, the sesquiterpene gene A0064, which could produce 10 possible sesquiterpenes in the heterologous expression system of A. oryzae, was knocked out to obtain A0064 knockout strain S. ochraceum (∆A0064). Steperoxide A could not be detected in S. ochraceum (∆A0064), demonstrating that A0064 was the only enzyme responsible for the biosynthesis of β-chamigrene (the sesquiterpene skeleton of steperoxide A) in S. ochraceum. This efficient system will enable precise targeting and multiplex editing of S. ochraceum genes, facilitating functional studies of genes involved in lignin degradation and natural product biosynthesis in S. ochraceum, and providing some valuable guidance for gene editing in tens of thousands of macrofungi.
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Gene editing in Plasmodium berghei made easy: Development of a CRISPR/Cas9 protocol using linear donor template and ribozymes for sgRNA generation.
Efficient reverse genetics approaches are critical for the study of many organisms. The CRISPR/Cas9 gene editing system has led to a plethora of new tools for geneticists. Here, we successfully established a simplified CRISPR/Cas9 system for the malaria model parasite Plasmodium berghei. The homologous directed repair (HDR) template is provided as a linear template with homologous arms of 600-700bp while the CRISPR elements sgRNA and Cas9 are encoded from a single plasmid utilizing the Ribozyme-Guide-Ribozyme (RGR) expression strategy. Our approach eliminates the need for negative selection markers since the plasmid cannot be incorporated into the genome. As a test case we inserted the FLAG encoding sequence into the ACT2 locus using this new approach. We showed that the genetic modification of this locus had no adverse effects on the completion of the P. berghei life cycle, including transmission through the mosquito.
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Taking responsibility, making irresponsibility: Controversies in human gene editing.
In current discussions, human germline editing is often called 'irresponsible'. Looking at the international summits on human gene editing held in 2015 and in 2018 and the announcement by He Jiankui of the birth of two gene-edited babies in November 2018, this article analyses how 'irresponsible' research was the result of various (dis)qualifications and demarcations. Against a background of discussions of responsibility, an individual scientist was singled out, his experiments were scrutinized for their soundness, legality and safety and ethical and moral stances were questioned. These are features of a process that I call 'irresponsibilization'. This irresponsibilization of research is entangled with calls for further action: Irresponsible research like that of He Jiankui should be contained, the veracity of knowledge claims needs to be confirmed, and institutions and decision-makers are called to act. The controversy turned 'irresponsible' into an active category, and rendered explicit its political, institutional and practical ramifications.
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Topoisomerase Inhibitors and PIM1 Kinase Inhibitors Improve Gene Editing Efficiency Mediated by CRISPR-Cas9 and Homology-Directed Repair.
The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system.
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