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Enhancing CRISPR/Cas systems with nanotechnology.
CRISPR/Cas systems have revolutionized biology and medicine, and have led to new paradigms in disease diagnostics and therapeutics. However, these complexes suffer from key limitations regarding barriers to cellular entry, stability in biological environments, and off-target effects. Integrating nanotechnology with CRISPR/Cas systems has emerged as a promising strategy to overcome these challenges and has further unlocked structures that accumulate preferentially in tissues of interest, have tunable pharmacological properties, and are activated in response to desired stimuli. Nanomaterials can also enhance CRISPR/Cas-mediated detection platforms by enabling faster, more sensitive, and convenient readouts. We highlight recent advances in this rapidly growing field. We also outline areas that need further development to fully realize the potential of CRISPR technologies.
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Generation of IL17RB Knockout Cell Lines Using CRISPR/Cas9-Based Genome Editing.
CRISPR/Cas9-based genome editing is an inexpensive and efficient tool for genetic modification. Here, we present a methodological approach for establishing interleukin-17 receptor B (IL17RB) knockout cell lines using CRISPR/Cas9-mediated genomic deletion. The IL17RB gene encodes for a cytokine receptor that specifically binds to IL17B and IL17E and is overexpressed in various cancers. The method involves CRISPR design, CRISPR cloning, delivery of the CRISPR clone into cells, and verification of IL17RB gene deletion by deletion screening primer design, genomic DNA extraction, and polymerase chain reaction (PCR). A similar approach can be used for generating mammalian cell lines with gene knockout for other genes of interest.
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Lonvoguran ziclumeran: a CRISPR-CAS9-based gene therapy for the treatment of hereditary angioedema.
INTRODUCTION: Hereditary angioedema (HAE) is a rare genetic disorder characterized by recurrent swelling caused by dysregulation of the kallikrein-kinin pathway. Although current therapies effectively reduce attack frequency, treatment remains lifelong. Lonvoguran ziclumeran (Lonvo-z; NTLA-2002) is the first systemically administered in vivo CRISPR/Cas9 gene-editing therapy designed to provide durable suppression of plasma kallikrein through permanent disruption of the KLKB1 gene. AREAS COVERED: This review summarizes the pathophysiology and current management of HAE, the development of Lonvo-z, its lipid nanoparticle delivery platform, and the technical advances enabling in vivo genome editing. Preclinical studies and clinical evidence, including early-phase trials and the Phase 3 HAELO study, are reviewed with emphasis on efficacy, safety and clinical implications. EXPERT OPINION: Lonvo-z represents a major milestone in precision medicine and the clinical application of systemic genome editing. A single administration has produced sustained reductions in plasma kallikrein levels and HAE attack frequency. Although long-term follow-up is ongoing, current evidence supports its potential as the first one-time disease-modifying treatment for HAE and a landmark advance in CRISPR-based therapeutics.
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Engineered Cas9 extracellular vesicles as a novel gene editing tool.
Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.
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Time Matters: Gene Editing at the Mouse 2-Cell Embryo Stage Boosts Knockin Efficiency.
Although endonuclease-mediated genome editing techniques offer significant improvement over traditional methods, they are still ineffective for introduction of large DNA sequences. Recently in Nature Biotechnology, Gu et al. (2018) developed a CRISPR-Cas strategy termed 2C-HR-CRISPR that generates fluorescent reporter tagging of genes with up to 95% knockin efficiency in mouse embryos.
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CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy.
The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previously untreatable genetic diseases. However, reports of spurious off-target editing by CRISPR/Cas9 pose a significant hurdle to realizing this potential. A deeper understanding of the factors that affect Cas9 specificity is vital for development of safe and efficient therapeutics. Here, we describe methods for the use of optical tweezers combined with confocal fluorescence microscopy and microfluidics for the analysis of on- and off-target activity of Cas9 activity.
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A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells.
Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3-6 weeks in order to understand genetic determinants of disease and precision medicine.
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CRISPR/Cas Genome Editing in Filamentous Fungi.
The review describes the CRISPR/CAS system and its adaptation for the genome editing in filamentous fungi commonly used for production of enzyme complexes, enzymes, secondary metabolites, and other compounds used in industrial biotechnology and agriculture. In the second part of this review, examples of the CRISPR/CAS technology application for improving properties of the industrial strains of fungi from the Trichoderma, Aspergillus, Penicillium, and other genera are presented. Particular attention is given to the efficiency of genome editing, as well as system optimization for specific industrial producers.
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Milestones of CRISPR/Cas9 system, promises and roadblocks.
In this issue of Cancer Treatment and Research Communications, Sarkar et al. describe CRISPR/Cas9-based gene-editing from its discovery to current views. Originally a bacterial adaptive immune system, CRISPR/Cas9 was adapted to mammalian cells as a tool to perform a wide range of actions given its capability of accurately targeting specific DNA loci. While the CRISPR/Cas9 system is readily used in laboratories as a research tool for a few years now, the first clinical trials have recently started utilizing this system in translational medicine. Although several obstacles have been resolved related to the usage of CRISPR/Cas9 on mammalian cells, the scientific community is facing new challenges with this technology.
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Multifunctional nano-polymer-based targeted delivery system for CRISPR/Cas9-Mediated hepatocellular carcinoma therapy.
CRISPR/Cas9 gene-editing technology exhibits substantial therapeutic potential for hepatocellular carcinoma (HCC); however, the targeted delivery of the CRISPR/Cas9 system into tumor cells remains a critical challenge requiring urgent exploration. Methyltransferase-Like 3 (METTL3), a key methyltransferase, drives HCC proliferation via multiple mechanisms. To address this challenge, a multifunctional delivery system was developed to efficiently deliver CRISPR/Cas9 plasmids targeting METTL3 (pMETTL3) into HCC cells. The cationic PEI, which facilitated the adsorption of pMETTL3 and protected it from lysosomal degradation, served as the polymeric backbone and was modified with deoxycholic acid (DOCA) to enhance its hydrophobicity. Meanwhile, lactobionic acid (LA) was grafted onto the structure to actively target HCC cells. The resulting pMETTL3/LPD was further functionalized with pH-sensitive and cleavable polyethylene glycol (PEG), aiming to reduce toxicity and enhance prolonged circulation. Results demonstrated that the delivery system maintains stability in physiological pH environments while achieving significantly enhanced accumulation in tumor tissues. Furthermore, the efficient cellular uptake of CRISPR/Cas9 plasmids enables precise gene editing, thereby effectively disrupting METTL3 expression, inducing apoptosis, and ultimately inhibiting HCC growth. This study presents a promising therapeutic strategy targeting METTL3 for HCC treatment and further expands the application of CRISPR/Cas9 gene-editing technology in cancer therapy.
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