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Rising Star Engineering the Genome for Curative Futures.
As a professor of biomedicine in the School of Life Sciences at East China Normal University (ECNU), I am dedicated to developing advanced genome editing technologies for disease modeling and precise gene therapy. My foundational training at Hunan Normal University and Texas A&M University cultivated a deep interest in using engineered cellular and animal models to understand human diseases. Since 2013, my laboratory at ECNU has pioneered the use of TALEN and CRISPR/Cas9 for the rapid generation of knockout mouse and rat models for disease studies. Once stepped in genome editing field, I shifted my focus to advancing editing tools and developing gene therapy strategies for genetic disorders and cancer. My team has developed a suite of high-performance base editors for nuclear DNA, mitochondrial DNA, and RNA, broadening editing capabilities while enhancing precision and safety. Leveraging these technologies, we have designed several therapeutic strategies that have shown efficacy in cellular and animal models of genetic diseases. Through collaborative efforts, we have successfully translated genome editing into clinical applications, contributing to the treatment of patients with β-thalassemia. Additionally, we have developed a non-viral, site-specific CAR-T platform for lymphoma therapy. Looking forward, I aim to develop the next generation of long-fragment, site-specific integration technologies and accelerate clinical translation to bring transformative cures to more patients.
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Topical gene editing therapeutics using lipid nanoparticles: 'gene creams' for genetic skin diseases?
Patients living with inherited skin diseases have benefited from recent advances in DNA sequencing technologies that provide new or improved diagnostics. However, developing and delivering new treatments for the 'genodermatoses' remains challenging. The goal of creating topical preparations that can recover the inherent gene pathology remains largely aspirational. However, recent progress in two fields - the chemistry of topical delivery formulations (lipid nanoparticles) and the molecular biology of gene repair (CRISPR-Cas9, base and prime editing) - presents new opportunities to address this unmet need. In this review, we discuss how lipid nanoparticle delivery vehicles could be used to deliver gene-editing tools to formulate topical 'gene creams' suitable for the treatment of genodermatoses. We summarize the historical landscape of topical therapeutics and advances in gene editing that may herald an era of new therapies for patients with inherited skin disorders.
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Targeted genome editing in a quail cell line using a customized CRISPR/Cas9 system.
Soon after RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease opened a new era of targeted genome editing, the CRISPR/Cas9 platform began to be extensively used to modify genes in various types of cells and organisms. However, successful CRISPR/Cas9-mediated insertion/deletion (indel) mutation remains to be demonstrated in avian cell lines. The objective of this study was to design a poultry-specific CRISPR/Cas9 system to efficiently introduce targeted deletion mutation in chromosomes of the quail muscle clone 7 (QM7) cell line using a customized quail CRISPR vector. In this study, two avian-specific promoters, quail 7SK (q7SK) promoter and CBh promoter, the hybrid form of cytomegalovirus and chicken β-actin promoters, were cloned into a CRISPR vector for the expression of guide RNA and Cas9 protein, respectively. Then, guide RNA, which was designed to target 20-base pair (bp) nucleotides in the quail melanophilin (MLPH) locus, was ligated to the modified CRISPR vector and transfected to QM7 cells. Our results showed multiple indel mutations in the quail MLPH locus in nearly half of the alleles being tested, suggesting the high efficiency of the system for targeted gene modification. The new CRISPR vector developed from this study has the potential application to generate knockout avian cell lines and knockout poultry.
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Spatial profiling of gene editing by in situ sequencing in mice and macaques.
Base and prime editing technologies precisely install defined nucleotide edits in both dividing and non-dividing cells, offering potential for correcting pathogenic mutations directly in organisms. However, to fully leverage their therapeutic potential, accurately measuring editing rates with high spatial resolution is crucial. Here we use imaging-based in situ sequencing (ISS) to map base and prime editing events within native tissues. We establish and validate this technology in mouse brains treated with intein-split adenine base editors or prime editors delivered via adeno-associated viral vectors. We next apply ISS in the liver of mice and macaques treated with adenine base editors encoded on lipid nanoparticle-encapsulated mRNA and guide RNA (RNA-LNP). Effective editing was observed across all metabolic zones of liver lobules. Moreover, in experiments where repeated doses of RNA-LNP are administered, the initial dose does not affect the editing efficiency and distribution of the subsequent dose. Our results demonstrate how ISS can visualize gene editing events in vivo and suggest that base editor delivery using RNA-LNP could be used to address a wide spectrum of metabolic liver diseases.
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CRISPR-Cas9 mediated understanding of plants' abiotic stress-responsive genes to combat changing climatic patterns.
Multiple abiotic stresses like extreme temperatures, water shortage, flooding, salinity, and exposure to heavy metals are confronted by crop plants with changing climatic patterns. Prolonged exposure to these adverse environmental conditions leads to stunted plant growth and development with significant yield loss in crops. CRISPR-Cas9 genome editing tool is being frequently employed to understand abiotic stress-responsive genes. Noteworthy improvements in CRISPR-Cas technology have been made over the years, including upgradation of Cas proteins fidelity and efficiency, optimization of transformation protocols for different crop species, base and prime editing, multiplex gene-targeting, transgene-free editing, and graft-based heritable CRISPR-Cas9 approaches. These developments helped to improve the knowledge of abiotic stress tolerance in crops that could potentially be utilized to develop knock-out varieties and over-expressed lines to tackle the adverse effects of altered climatic patterns. This review summarizes the mechanistic understanding of heat, drought, salinity, and metal stress-responsive genes characterized so far using CRISPR-Cas9 and provides data on potential candidate genes that can be exploited by modern-day biotechnological tools to develop transgene-free genome-edited crops with better climate adaptability. Furthermore, the importance of early-maturing crop varieties to withstand abiotic stresses is also discussed in this review.
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CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis.
The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.
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Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3'-overhang.
Genome editing has been harnessed through the development of CRISPR system, and the CRISPR from Prevotella and Francisella 1 (Cpf1) system has emerged as a promising alternative to CRISPR-Cas9 for use in various circumstances. Despite the inherent multiple advantages of Cpf1 over Cas9, the adoption of Cpf1 has been unsatisfactory because of target-dependent insufficient indel efficiencies. Here, we report an engineered CRISPR RNA (crRNA) for highly efficient genome editing by Cpf1, which includes a 20-base target-complementary sequence and a uridinylate-rich 3'-overhang. When the crRNA is transcriptionally produced, crRNA with a 20-base target-complementary sequence plus a U4AU4 3'-overhang is the optimal configuration. U-rich crRNA also maximizes the utility of the AsCpf1 mutants and multiplexing genome editing using mRNA as the source of multiple crRNAs. Furthermore, U-rich crRNA enables a highly safe and specific genome editing using Cpf1 in human cells, contributing to the enhancement of a genome-editing toolbox.
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CRISPR-Cas9 mediated enhancement of abiotic stress resilience in tomato: a comprehensive review of target genes.
Tomato (Solanum lycopersicum L.), a major vegetable crop grown worldwide, is consumed as both fresh and processed products. Concerns about the impact of abiotic stresses on tomato production are growing worldwide as climate change alters global weather patterns, adversely affecting crop yield and produce quality due to stresses like salt, heat, cold, and drought. Conventional breeding approaches such as hybridization, marker-assisted selection (MAS), and mutation breeding, have long been utilized to improve tomato resilience against abiotic stresses. These approaches are often hampered by extensive field trials, and require multiple generations limiting their efficiency in rapidly developing stress-tolerant cultivars. The efforts of traditional breeding systems are hindered by the narrow genetic base of tomatoes which poses a major bottleneck. Researchers have utilized CRISPR-Cas genome-editing technology to address this challenge to offer a precise and accelerated alternative for enhancing stress resilience in tomato. This versatile tool has gained attention for its simple, precise, and effective gene-editing capabilities. CRISPR-Cas based genome editing has successfully modified key genes related to stress-response pathways, enhancing abiotic stress resilience. Developing resistant cultivars help mitigate the impact of abiotic stress, thereby contributing to increased food production and food security. This review highlights recent progress in use of CRISPR-Cas9 gene editing to enhance tomato resilience to abiotic stresses.
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Insights into pegRNA design from editing of the cardiomyopathy-associated phospholamban R14del mutation.
Prime editing (PE) represents a transformative genome-editing technology and enables precise insertions, deletions, and base substitutions without introducing double-strand breaks, thereby reducing undesired indels and off-target effects. Despite advancements in enhanced prime editors and optimized prime editing guide RNAs (pegRNAs), designing effective pegRNAs remains a major challenge. The phospholamban (PLN) R14del mutation is associated with cardiomyopathies, making it a crucial target for precise gene-editing strategies. In this study, we explored pegRNA features that contribute to high editing efficiency using the FluoPEER.PLN R14del reporter cell line. Through systematic screening, we identified three pegRNAs with significantly enhanced editing efficiency. Our findings underscore the importance of pegRNA secondary structure and stability in optimizing prime editing, providing valuable insights into precise gene correction strategies.
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From Sequencing to Genome Editing for Cotton Improvement.
Traditional breeding techniques are proven, but additional knowledge learned from genome sequencing provides vast new data that might help identify gene targets for improving cotton sustainability. CRISPR/Cas9 provides a powerful tool for precision cotton breeding. Here, we discuss the opportunities and challenges of genome sequencing and editing for cotton improvement.
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