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Protocol for enhancing CRISPR-Cas9 genome editing using histone deacetylase inhibition and engineered virus-like particle delivery.
We present a 10-fold faster, accurate, and more efficient (FAME)-CRISPR-Cas9 gene editing workflow utilizing histone deacetylase inhibitor (HDACi)-mediated chromatin relaxation and engineered virus-like particle (eVLP) delivery of Cas9. We describe steps for optimizing HDACi concentration, euchromatinization timing, and Cas9 delivery/expression to improve CRISPR-Cas9 editing efficiency and efficacy. This protocol can eliminate the need for single-cell cloning and reduce experimental timelines up to 10-fold while minimizing HDACi-mediated toxicity. For complete details on the use and execution of this protocol, please refer to Djamshidi et al.1.
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Guide RNA scaffold variants enabled easy cloning of large gRNA cluster for multiplexed gene editing.
Cas9 protein-mediated gene editing has revolutionized genetic manipulation in most organisms. There are many cases where multiplexed gene editing is needed. Cas9 is capable of multiplex gene editing when expressed with multiple guide RNAs. Conventional cloning methods for multiplexed gene editing vector is not efficient due to repeated use of a single-guide RNA scaffold and inefficient ligation. In this study, we conducted structure-guided mutagenesis and random mutagenesis on the original sgRNA scaffold and identified a large number of functional sgRNA scaffold variants. With these scaffold variants and different tRNAs, fusion polymerase chain reaction protocol was developed to rapidly synthesize spacer-scaffold-tRNA-spacer units with up to 9 targets. In conjunction with golden gate cloning, gene editing vectors with up to 24 target sites were efficiently cloned in one-step cloning. One such gene editing vector targeting 12 genes in tomato were tested in stable transformation and 10 out of the 12 genes were found mutated in a single transgenic line. To facilitate the application of multiplexed gene editing using these scaffold variants and tRNAs from different species, a webserver was created to generate primer sets and provide template sequences for the synthesis of large sgRNA expression units based on the user-supplied target sequences and species.
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Advanced nanotheranostic approaches for targeted glioblastoma treatment: a synergistic fusion of CRISPR-Cas gene editing, AI-driven tumor profiling, and BBB-modulation.
Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. It is hard to treat because it is very invasive, has a lot of genetic variation, and the blood-brain barrier (BBB) limits its growth. Traditional GBM treatments, including surgery, radiation and chemotherapy have only marginally improved survival requiring a paradigm shift. This review starts a new way of thinking about how to treat GBM by combining multi-responsive nanotheranostics, next-generation genome engineering, and AI-enabled stratification. The study compares smart nanocarriers that can transport payloads accurately and only when needed. CRISPR/Cas systems are useful for directly editing oncogenes, controlling GBM signaling cascades, making resistant cells sensitive again, and changing the ways that cells avoid the immune system. Nanoparticle-mediated CRISPR delivery and BBB disruption approaches, including targeted ultrasound, receptor-mediated transcytosis, and exosome-based delivery, could help get around the neuroprotective shield without damaging it. This review investigates the molecular aspects of the BBB, highlighting its dual role as a metabolic and efflux transporter-mediated barrier, and assesses cutting-edge nanocarrier systems, in particular hybrid exosome-nanoparticle designs, that aim to circumvent these limitations. Additionally, it explores the current limitations and possible future applications of gene editing tools and AI models for navigating the genomic instability and ever-changing tumor microenvironment of GBM. This study also discusses the ethical and legal issues related to these developing technologies, therefore stressing the need for clinically feasible, ethically controlled innovation pipelines. With an emphasis on cutting-edge research, this review delves into emerging areas such as nanotheranostics, cellular composition of GBM, CRISPR-based gene editing, AI-enabled precision medicine, and targeted disruption of the blood-brain barrier.
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[Bacterial genome editing using the CRISPR/Cas12a system].
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Silkworm mutagenesis using a ribonucleoprotein-based CRISPR/Cas12a system.
The development of highly efficient genome editing tools has revolutionized developmental biology and genetic studies in silkworm. Although methods based on CRISPR/Cas9 are currently popular, the Cas12a system has emerged as a promising option. However, it has not yet been applied to target the silkworm genome in vivo, and its activity in silkworm has not yet been characterized. In this study, we established a ribonucleoprotein-based CRISPR/Cas12a system, and compared it to the CRISPR/Cas9 system using 19 crRNA and 17 sgRNAs to target three different genes in vivo. Although Cas12a generates mutants less efficiently than Cas9, we used it successfully to generate transmissible indels, and demonstrated its application by targeting the FibH and mp genes to produce mutants with the expected phenotypes. We also assessed the influence of temperature (37 °C vs. 25 °C) on Cas12a activity, and demonstrated that the effects are target dependent. In summary, we have established a ribonucleoprotein-based CRISPR/Cas12a system in silkworm that offers a practical alternative to CRISPR/Cas9 and extends the genome editing tool box available for silkworm research.
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Gene Editing for Corneal Stromal Regeneration.
CRISPR/Cas9 gene editing holds the promise of sequence-specific alteration of the genome to achieve therapeutic benefit in the treated tissue. Cas9 is an RNA-guided nuclease in which the sequence of the RNA can be altered to match the desired target. However, care must be taken in target choice and RNA guide design to ensure both maximum on-target and minimum off-target activity. The cornea is an ideal tissue for gene therapy due to its small surface area, accessibility, immune privilege, avascularity, and ease of visualization. Herein, we describe the design, testing, and delivery of Cas9 and guide RNAs to target genes expressed in the cornea.
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CRISPR-Cas9 assisted functional gene editing in the mushroom Ganoderma lucidum.
The genetic manipulation of basidiomycete mushrooms is notoriously difficult and immature, and there is a lack of research reports on clustered regularly interspaced short palindromic repeat (CRISPR) based gene editing of functional genes in mushrooms. In this work, Ganoderma lucidum, a famous traditional medicinal basidiomycete mushroom, which produces a type of unique triterpenoid-anti-tumor ganoderic acids (GAs), was used, and a CRISPR/CRISPR-associated protein-9 nuclease (Cas9) editing system for functional genes of GA biosynthesis was constructed in the mushroom. As proof of concept, the effect of different gRNA constructs with endogenous u6 promoter and self-cleaving ribozyme HDV on ura3 disruption efficiency was investigated at first. The established system was applied to edit a cytochrome P450 monooxygenase (CYP450) gene cyp5150l8, which is responsible for a three-step biotransformation of lanosterol at C-26 to ganoderic acid 3-hydroxy-lanosta-8, 24-dien-26 oic acid. As a result, precisely edited cyp5150l8 disruptants were obtained after sequencing confirmation. The fermentation products of the wild type (WT) and cyp5150l8 disruptant were analyzed, and a significant decrease in the titer of four identified GAs was found in the mutant compared to WT. Another CYP gene involved in the biosynthesis of squalene-type triterpenoid 2, 3; 22, 23-squalene dioxide, cyp505d13, was also disrupted using the established CRISPR-Cas9 based gene editing platform of G. lucidum. The work will be helpful to strain molecular breeding and biotechnological applications of G. lucidum and other basidiomycete mushrooms.
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CRISPR/Cas9-based gene-editing technology for sickle cell disease.
Sickle cell disease (SCD) is the most common monogenic hematologic disorder and is essentially congenital hemolytic anemia caused by an inherited point mutation in the β-globin on chromosome 11. Although the genetic basis of SCD was revealed as early as 1957, treatment options for SCD have been very limited to date. Hematopoietic stem cell transplantation (HSCT) was thought to hold promise as a cure for SCD, but the available donors were still only 15% useful. Gene therapy has advanced rapidly into the 21st century with the promise of a cure for SCD, and gene editing strategies based on the cluster-based regularly interspaced short palindromic repeat sequence (CRISPR)/Cas9 system have revolutionized the field of gene therapy by precisely targeting genes. In this paper, we review the pathogenesis and therapeutic approaches of SCD, briefly summarize the delivery strategies of CRISPR/Cas9, and finally discuss in depth the current status, application barriers, and solution directions of CRISPR/Cas9 in SCD. Through the review in this paper, we hope to provide some references for gene therapy in SCD.
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Personalised genome editing - The future for corneal dystrophies.
The potential of personalised genome editing reaching the clinic has come to light due to advancements in the field of gene editing, namely the development of CRISPR/Cas9. The different mechanisms of repair used to resolve the double strand breaks (DSBs) mediated by Cas9 allow targeting of a wide range of disease causing mutations. Collectively, the corneal dystrophies offer an ideal platform for personalised genome editing; the majority of corneal dystrophies are monogenic, highly penetrant diseases with a known pattern of inheritance. This genetic background coupled with the accessibility, ease of visualisation and immune privilege status of the cornea make a gene editing strategy for the treatment of corneal dystrophies an attractive option. Off-target cleavage is a major concern for the therapeutic use of CRISPR/Cas9, thus current efforts in the gene editing field are focused on improving the genome-wide specificity of Cas9 to minimise the risk of off-target events. In addition, the delivery of CRISPR/Cas9 to different tissues is a key focus; various viral and non-viral platforms are being explored to develop a vehicle that is highly efficient, specific and non-toxic. The rapid pace and enthusiasm with which CRISPR/Cas9 has taken over biomedical research has ensured the personalised medicine revolution has been realised. CRISPR/Cas9 has recently been utilised in the first wave of clinical trials, and the potential for a genome editing therapy to treat corneal dystrophies looks promising. This review will discuss the current status of therapeutic gene editing in relation to the corneal dystrophies.
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Stem Cell Gene Editing Improved Sickle Cell Disease Outcomes.
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