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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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The Synergy between CRISPR and Chemical Engineering.
Gene therapy and transgenic research have advanced quickly in recent years due to the development of CRISPR technology. The rapid development of CRISPR technology has been largely benefited by chemical engineering. Firstly, chemical or synthetic substance enables spatiotemporal and conditional control of Cas9 or dCas9 activities. It prevents the leaky expression of CRISPR components, as well as minimizes toxicity and off-target effects. Multi-input logic operations and complex genetic circuits can also be implemented via multiplexed and orthogonal regulation of target genes. Secondly, rational chemical modifications to the sgRNA enhance gene editing efficiency and specificity by improving sgRNA stability and binding affinity to on-target genomic loci, and hence reducing off-target mismatches and systemic immunogenicity. Chemically-modified Cas9 mRNA is also more active and less immunogenic than the native mRNA. Thirdly, nonviral vehicles can circumvent the challenges associated with viral packaging and production through the delivery of Cas9-sgRNA ribonucleoprotein complex or large Cas9 expression plasmids. Multi-functional nanovectors enhance genome editing in vivo by overcoming multiple physiological barriers, enabling ligand-targeted cellular uptake, and blood-brain barrier crossing. Chemical engineering can also facilitate viral-based delivery by improving vector internalization, allowing tissue-specific transgene expression, and preventing inactivation of the viral vectors in vivo. This review aims to discuss how chemical engineering has helped improve existing CRISPR applications and enable new technologies for biomedical research. The usefulness, advantages, and molecular action for each chemical engineering approach are also highlighted.
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The Bibliometric Landscape of Gene Editing Innovation and Regulation in the Worldwide.
Gene editing (GE) has become one of the mainstream bioengineering technologies over the past two decades, mainly fueled by the rapid development of the CRISPR/Cas system since 2012. To date, plenty of articles related to the progress and applications of GE have been published globally, but the objective, quantitative and comprehensive investigations of them are relatively few. Here, 13,980 research articles and reviews published since 1999 were collected by using GE-related queries in the Web of Science. We used bibliometric analysis to investigate the competitiveness and cooperation of leading countries, influential affiliations, and prolific authors. Text clustering methods were used to assess technical trends and research hotspots dynamically. The global application status and regulatory framework were also summarized. This analysis illustrates the bottleneck of the GE innovation and provides insights into the future trajectory of development and application of the technology in various fields, which will be helpful for the popularization of gene editing technology.
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Lethal endotoxin (ccdB) based counterselection improved the efficiency of sequential gene editing in Escherichia coli.
The CRISPR/Cas9-based technology has been used for sequential gene editing in E. coli. The plasmids carrying the sgRNA and/or Cas9 genes need to be cured after each round of editing. Curing of these plasmids, particularly the sgRNA plasmid, limits the efficiency of sequential gene editing. In this study, a lethal endotoxin (ccdB) based counterselection was established for improving the overall efficiency of sequential gene editing in E. coli. This approach was validated for sequential editing (deletion) of cstA and ppsA genes in HBUT-P2 strain (W derivative). The experimental results showed that the transformation efficiency of sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20) reached 108-109 cfu/μg-DNA, resulting in a 100% and 93.75% recombination rate for cstA and ppsA gene, respectively. Upon completion of cstA gene editing, the sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (cstA)) was effectively cured through ccdB based counterselection at 42 °C, with a 43.75% efficiency. At the end of sequential editing of ppsA gene, both Cas9 (25A) and sgRNA (pTargetF-tcr-PL-ccdB-N20 (ppsA)) plasmids were cured simultaneously through the sacB and ccdB based counterselections by incubating the cells on LB-sucrose (5%) plate at 42 °C, achieving a curing rate of 100% for Cas9 plasmid (25A), 37.5% for sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (ppsA)), and 37.5% for both Cas9 and sgRNA plasmids. Moreover, this approach was further validated through efficient site-specific insertion of the csc operon into the slmA gene in DH5α (K12 derivative) and S322 (C derivative) strains. These results demonstrated that the endotoxin (ccdB) based counterselection improved the transformation efficiency of sgRNA plasmid, the recombination rate of the editing target gene, the curing rate of sgRNA plasmid, and the overall efficiency of sequential gene editing.
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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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Xenotransplantation: Promise, progress, and risks.
Xenotransplantation has seen remarkable progress in recent years, largely driven by the advent of CRISPR-Cas9 gene editing technology. Several patients have received genetically modified kidney and heart transplants, achieving graft and patient survival ranging from days to months. This review offers a brief history of xenotransplantation and highlights advancements in gene editing that represent breakthroughs in minimizing rejection. It also presents recent cases of xenotransplantation and their clinical outcomes. Finally, the review addresses the ethical considerations and risks associated with this emerging field.
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Harnessing Gene Editing Technology for Tumor Microenvironment Modulation: An Emerging Anticancer Strategy.
Cancer is a multifaceted disease influenced by both intrinsic cellular traits and extrinsic factors, with the tumor microenvironment (TME) being crucial for cancer progression. To satisfy their high proliferation and aggressiveness, cancer cells always plunder large amounts of nutrients and release various signals to their surroundings, forming a dynamic TME with special metabolic, immune, microbial and physical characteristics. Due to the neglect of interactions between tumor cells and the TME, traditional cancer therapies often struggle with challenges such as drug resistance, low efficacy, and recurrence. Importantly, the development of gene editing technologies, particularly the CRISPR-Cas system, offers promising new strategies for cancer treatment. Combined with nanomaterial strategies, CRISPR-Cas technology exhibits precision, affordability, and user-friendliness with reduced side effects, which holds great promise for profoundly altering the TME at the genetic level, potentially leading to lasting anticancer outcomes. This review will delve into how CRISPR-Cas can be leveraged to manipulate the TME, examining its potential as a transformative anticancer therapy.
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[Bacterial genome editing using the CRISPR/Cas12a system].
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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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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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