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The current state of Cytotherapy and the field of cell and gene therapy.
2024 marked a transformative phase for cell and gene therapy (CGT) with significant advancements in scientific innovation, regulatory approvals and commercialization milestones. This review highlights key developments in CGT, including innovations in chimeric antigen receptor (CAR)-T therapies, mesenchymal stromal cells (MSCs), gene editing and regenerative medicine, alongside challenges in scalability, regulation and safety. Prominent breakthroughs in CAR-T technology extended its applications beyond oncology to autoimmune diseases, including lupus and systemic sclerosis. Gene therapies achieved major milestones, exemplified by regulatory approval for the treatment of hemophilia, sickle cell disease and other genetic diseases. Further advancements in delivery systems, including lipid nanoparticles and engineered viral vectors were achieved. Refinements in clustered regularly interspaced short palindromic repeats-Cas9 and base-editing tools improved precision and reduced off-target effects, enabling new approaches for genetic disorders. Global collaboration underscored the collective effort to accelerate CGT progress. MSCs remain central to CGT research, focusing on their immunomodulatory properties and clinical applications in autoimmune diseases and graft-versus-host disease. Economic and policy landscapes evolved alongside scientific advancements. Record-breaking approvals and biotech IPOs underscored CGT's economic potential, while affordability and equitable access emerged as critical challenges. Regulatory agencies advanced harmonized guidelines for manufacturing and clinical evaluation, streamlining global access to these therapies. Ethical considerations, including the affordability of therapies and the need for diverse clinical trial representation, remained prominent. Despite progress, challenges persist in scalability, safety and regulatory harmonization. Manufacturing improvements are essential to meet growing demand, while addressing safety concerns, such as off-target gene-editing effects and tumorigenicity in MSC therapies, remains paramount.
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Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing has become a promising approach for efficient and versatile genetic engineering in various organisms; however, simple and precise nucleotide modification methods in filamentous fungi have been restricted to double crossover type homologous recombination (HR). In this study, we developed a novel genome editing strategy via single crossover-mediated HR in the model filamentous fungus Pyricularia (Magnaporthe) oryzae. This method includes the CRISPR/Cas9 system and a donor vector harboring a single homology arm with point mutations at the CRISPR/Cas9 cleavage site. Using this strategy, we demonstrated highly efficient and freely programmable base substitutions within the desired genomic locus, and target gene disrupted mutants were also obtained via a shortened (100-1000 bp) single homology arm. We further demonstrated that this method allowed a one-step GFP gene knock-in at the C-terminus of the targeted gene. Since the genomic recombination does not require an intact protospacer-adjacent motif within the donor construct and any additional modifications of host components, this method can be used in various filamentous fungi for CRISPR/Cas9-based basic and applied biological analyses.
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RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?
Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects.
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CRISPR-Cas systems of lactic acid bacteria and applications in food science.
CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) systems are widely distributed in lactic acid bacteria (LAB), contributing to their RNA-mediated adaptive defense immunity. The CRISPR-Cas-based genetic tools have exhibited powerful capability. It has been highly utilized in different organisms, accelerating the development of life science. The review summarized the components, adaptive immunity mechanisms, and classification of CRISPR-Cas systems; analyzed the distribution and characteristics of CRISPR-Cas system in LAB. The review focuses on the development of CRISPR-Cas-based genetic tools in LAB for providing latest development and future trend. The diverse and broad applications of CRISPR-Cas systems in food/probiotic industry are introduced. LAB harbor a plenty of CRISPR-Cas systems, which contribute to generate safer and more robust strains with increased resistance against bacteriophage and prevent the dissemination of plasmids carrying antibiotic-resistance markers. Furthermore, the CRISPR-Cas system from LAB could be used to exploit novel, flexible, programmable genome editing tools of native host and other organisms, resolving the limitation of genetic operation of some LAB species, increasing the important biological functions of probiotics, improving the adaptation of probiotics in complex environments, and inhibiting the growth of foodborne pathogens. The development of the genetic tools based on CRISPR-Cas system in LAB, especially the endogenous CRISPR-Cas system, will open new avenues for precise regulation, rational design, and flexible application of LAB.
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CRISPR/Cas9 for the Clinician: Current uses of gene editing and applications for new therapeutics in oncology.
Precise genomic editing has given rise to treatments in previously untreatable genetic diseases and has led to revolutions in treatment for cancer. In the past decade, the discovery and development of clustered regularly interspaced short palindromic repeats (CRISPR) technologies has led to advances across medicine and biotechnology. Specifically, the CRISPR/Cas9 system has improved translational discovery and therapeutics for oncology across tumor types. In this review, we briefly summarize the history and development of CRISPR, explain CRISPR-Cas systems and CRISPR gene editing tools, highlight the development and application of CRISPR technologies for translational and therapeutic purposes in different oncologic tumors, and review novel treatment paradigms using CRISPR in immuno-oncology, including checkpoint inhibitors and chimeric antigen receptor T cell therapy.
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QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii.
Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.
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CRISPR/Cas genome editing to optimize pharmacologically active plant natural products.
Since time immemorial, human use medicinal plants as sources of food, therapy and industrial purpose. Classical biotechnology and recent next-generation sequencing (NGS) techniques have been successfully used to optimize plant-derived natural-products of biomedical significance. Earlier, protein based editing tools viz. zinc-finger nucleases (ZFNs) and transcription activator-like endonucleases (TALENs) have been popularized for transcriptional level genome manipulation. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease system is an efficient, robust and selective site-directed mutagenesis strategy for RNA-guided genome-editing. CRISPR/Cas9 genome-editing tool employs designed guide-RNAs that identifies a 3 base-pair protospacer adjacent motif (PAM) sequence occurring downstream of the target-DNA. The present review comprehensively complies the recent literature (2010-2020) retrieved from scientific-databases on the application of CRISPR/Cas9-editing-tools as potent genome-editing strategies in medicinal-plants discussing the recent developments, challenges and future-perspectives with notes on broader applicability of the technique in plants and lower-organisms. In plants, CRISPR/Cas-editing has been implemented successfully in relation to crop-yield and stress-tolerance. However, very few medicinal plants have been edited using CRISPR/Cas genome tool owing to the lack of whole-genome and mRNA-sequences and shortfall of suitable transformation and regeneration strategies. However, recently a number of plant secondary metabolic-pathways (viz. alkaloid, terpenoid, flavonoids, phenolic, saponin etc.) have been engineered employing CRISPR/Cas-editing via knock-out, knock-in, point-mutation, fine-tuning of gene-expression and targeted-mutagenesis. This genome-editing tool further extends its applicability incorporating the tools of synthetic- and systems-biology, functional-genomics and NGS to produce genetically-engineered medicinal-crops with advanced-traits facilitating the production of pharmaceuticals and nutraceuticals.
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A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice.
BACKGROUND: Microinjection of clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-related RNA and DNA into fertilized eggs is a novel approach for creating gene-modified mice. Blastocysts obtained just before implantation may be appropriate for testing the fidelity of CRIPSR/Cas9-mediated genome editing because they can be individually handled in vitro and obtained 3days after microinjection, thus allowing researchers to check mutations rapidly. However, it is not known whether indel mutations caused by the CRISPR/Cas9 system can be reproducibly detected in embryos. In this study, we assessed the detection of CRISPR/Cas9-induced mutations in embryos. RESULTS: T7 endonuclease I was more effective than Surveyor nuclease for detecting mutations in annealed fragments derived from 2 plasmids, which contained nearly identical sequences. Mouse fertilized eggs were microinjected with CRISPR/Cas9-related RNA/DNA to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor (G protein-coupled) activity modifying protein 2 (Ramp2) gene. Individual blastocysts were lysed to obtain crude DNA solutions, which were used for polymerase chain reaction (PCR) assays. T7 endonuclease I-based PCR and sequencing analysis demonstrated that 25-100% of the embryos were knockout embryos and 7-57% of the embryos were knockin embryos. Our results also established that crude DNA from a single blastocyst was an appropriate template for Whole genome amplification and subsequent assessment by PCR and the T7 endonuclease I-based assay. CONCLUSIONS: The single blastocyst-based assay was useful for determining whether CRISPR/Cas9-mediated genome editing worked in murine embryos.
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CRISPR-based epigenome editing: mechanisms and applications.
Epigenomic anomalies contribute significantly to the development of numerous human disorders. The development of epigenetic research tools is essential for understanding how epigenetic marks contribute to gene expression. A gene-editing technique known as CRISPR (clustered regularly interspaced short palindromic repeats) typically targets a particular DNA sequence using a guide RNA (gRNA). CRISPR/Cas9 technology has been remodeled for epigenome editing by generating a 'dead' Cas9 protein (dCas9) that lacks nuclease activity and juxtaposing it with an epigenetic effector domain. Based on fusion partners of dCas9, a specific epigenetic state can be achieved. CRISPR-based epigenome editing has widespread application in drug screening, cancer treatment and regenerative medicine. This paper discusses the tools developed for CRISPR-based epigenome editing and their applications.
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Multiple homologous genes knockout (KO) by CRISPR/Cas9 system in rabbit.
The CRISPR/Cas9 system is a highly efficient and convenient genome editing tool, which has been widely used for single or multiple gene mutation in a variety of organisms. Disruption of multiple homologous genes, which have similar DNA sequences and gene function, is required for the study of the desired phenotype. In this study, to test whether the CRISPR/Cas9 system works on the mutation of multiple homologous genes, a single guide RNA (sgRNA) targeting three fucosyltransferases encoding genes (FUT1, FUT2 and SEC1) was designed. As expected, triple gene mutation of FUT1, FUT2 and SEC1 could be achieved simultaneously via a sgRNA mediated CRISPR/Cas9 system. Besides, significantly reduced serum fucosyltransferases enzymes activity was also determined in those triple gene mutation rabbits. Thus, we provide the first evidence that multiple homologous genes knockout (KO) could be achieved efficiently by a sgRNA mediated CRISPR/Cas9 system in mammals, which could facilitate the genotype to phenotype studies of homologous genes in future.
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