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A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha.
The liverwort Marchantia polymorpha is a widely used model organism for studying land-plant biology, and it has also proven to be a promising testbed for bioengineering. CRISPR/Cas9 technology has become a transformative tool for precise genome modifications in M. polymorpha; however, a robust method for the simultaneous expression of multiple gRNAs, which is crucial for enhancing the versatility of CRISPR/Cas9-based genome editing, has yet to be fully developed. In this study, we introduce an adaptation from the OpenPlant kit CRISPR/Cas9 tools that facilitates expression of multiple gRNAs from a single transcript through incorporation of tRNA sequences. The ability to deliver multiple gRNAs simultaneously significantly improves the capacity and scalability of genome editing in M. polymorpha. Additionally, by combining this vector system with a simplified and optimized protocol for thallus transformation, we further streamline the generation of CRISPR/Cas9 mutants in M. polymorpha. The resulting gene-editing system offers a multipurpose, time-saving, and straightforward tool for advancing functional genomics in M. polymorpha, enabling more comprehensive genetic modifications and genome engineering.
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New CRISPR Technology for Creating Cell Models of Lipoprotein Assembly and Secretion.
PURPOSE OF REVIEW: This review is aimed at providing an overview of new developments in gene editing technology, including examples of how this technology has been used to develop cell models for studying the effects of gene ablation or missense mutations on lipoprotein assembly and secretion. RECENT FINDINGS: CRISPR/Cas9-mediated gene editing is superior to other technologies because of its ease, sensitivity, and low off-target effects. This technology has been used to study the importance of microsomal triglyceride transfer protein in the assembly and secretion of apolipoprotein B-containing lipoproteins, as well as to establish causal effects of APOB gene missense mutations on lipoprotein assembly and secretion. CRISPR/Cas9 technology is anticipated to provide unprecedented flexibility in studying protein structure and function in cells and animals and to yield mechanistic insights into variants in the human genome.
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What now for human genome editing?
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Gene Editing for Treatment and Prevention of Human Diseases: A Global Survey of Gene Editing-Related Researchers.
In the next decades, gene editing technologies are expected to be used in the treatment and prevention of human diseases. Yet, the future uses of gene editing in medicine are still unknown, including its applicability and effectiveness to the treatment and prevention of infectious diseases, cancer, and monogenic and polygenic hereditary diseases. This study aims to address this gap by analyzing the views of over 1,000 gene editing-related researchers from all over the world. Some of our survey results show that, in the next 10 years, DNA double-strand breaks are expected to be the main method for gene editing, and CRISPR-Cas systems to be the mainstream programmable nuclease. In the same period, gene editing is expected to have more applicability and effectiveness to treat and prevent infectious diseases and cancer. Off-targeting mutations, reaching therapeutic levels of editing efficiency, difficulties in targeting specific tissues in vivo, and regulatory and ethical challenges are among the most relevant factors that might hamper the use of gene editing in humans. In conclusion, our results suggest that gene editing might become a reality to the treatment and prevention of a variety of human diseases in the coming 10 years. If the future confirms these researchers' expectations, gene editing could change the way medicine, health systems, and public health deal with the treatment and prevention of human diseases.
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In utero gene editing and precision medicine.
Advances in preconception and prenatal genetic testing, in combination with the rapid evolution and regulatory acceptance of targeted gene therapy technologies, has unveiled promising opportunities for the antenatal treatment of genetic disease by way of in utero gene editing and precision medicine. Recent regulatory guidance, put forth by the Federal Drug Administration (FDA), on gene editing has the exciting potential to accelerate the development and implementation of therapeutics for rare and ultrarare diseases. In this review, we highlight recent advances that have occurred in the fields of in utero gene editing and precision medicine and the opportune regulatory landscape that may facilitate the clinical translation of therapeutics in the future. We additionally review the indispensable role of prenatal genetic diagnosis, including chorionic villus sampling, amniocentesis, and noninvasive prenatal testing, as the diagnostic gateway through which candidates for in utero gene and precision therapies will be identified.
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A simplified and improved protocol of rice transformation to cater wide range of rice cultivars.
The latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efficiencies which necessities screening of large number of transgenic plants to find the suitable edits. In the present study, we have simplified the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76-78 days. We also obtained about 90% regeneration efficiency for both Japonica and Indica cultivars. The transformation efficiency was about 97% in the case of Japonica and 69-83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efficiency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efficiency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars.
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Crispr/Cas9 homologous recombination (HR).
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Implementing CRISPR-Cas12a for Efficient Genome Editing in Yarrowia lipolytica.
The oleaginous yeast Yarrowia lipolytica has emerged as an industrially relevant chassis to produce various valuable chemicals. Metabolic engineering of Y. lipolytica relies on the availability of genetic engineering tools. Existing engineering strategies for this yeast include homologous recombination, random integration, and episomal plasmid-based gene expression. CRISPR-Cas9 based genome-editing toolbox has also been developed to facilitate multiplexed gene disruption and regulation. Alternative to Cas9, the CRISPR effector Cas12a has also been adopted to perform genome engineering in multiple species. Due to its distinctive features such as short and simple crRNA structure, the ability to process its own crRNA and T-rich PAM sequence (TTTN), Cas12a holds promising potential to be developed as an efficient genome-editing tool. In this chapter, we describe the protocol to implement multiplexed genome editing in Y. lipolytica. The delivery of AsCas12a and crRNA expression via a single plasmid was described. CRISPR-Cas12a-based genome editing could expand the genetic toolbox of Y. lipolytica, whihc is complementary to the classical Cas9-based tools.
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Facilitating Gene Editing in Human Lymphoma Cells Using Murine Ecotropic γ-Retroviruses.
Genetic modifications using CRISPR-Cas9 have revolutionized cancer research and other preclinical studies. Exceptionally, these efficient tools are inadequate in a few disease models and cell lines due to the aberrant differentiation states and the accumulation of excessive somatic mutations that compromise the robustness of viral gene delivery and stable transduction. A couple of B lymphoma cell lines fall into this category where lentiviral transfection becomes inefficient and exhibits variable efficiency. Additionally, lentiviral delivery requires high biosafety levels. To address this challenge, we have developed a two-step strategy that supports CRISPR-Cas9 through lentivirus and murine ecotropic γ-retrovirus. By engineering B lymphoma cell lines to express Cas9 and mCat1, a specific receptor for ecotropic retroviruses, we enable efficient and safe gene editing through ecotropic γ-retrovirus. We demonstrate the efficacy of this method by generating IgM-deficient B lymphoma cell lines. This innovative approach simplifies protocols, enhances accessibility, and paves the way for standardized gene manipulation of B cell lymphoma models for molecular cell biology research.
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Non-viral vector-based genome editing for cancer immunotherapy.
Despite the exciting promise of cancer immunotherapy in the clinic, immune checkpoint blockade therapy and T cell-based therapies are often associated with low response rates, intrinsic and adaptive immune resistance, and systemic side effects. CRISPR-Cas-based genome editing appears to be an effective strategy to overcome these unmet clinical needs. As a safer delivery platform for the CRISPR-Cas system, non-viral nanoformulations have been recently explored to target tumor cells and immune cells, aiming to improve cancer immunotherapy on a gene level. In this review, we summarized the efforts of non-viral vector-based CRISPR-Cas-mediated genome editing in tumor cells and immune cells for cancer immunotherapy. Their design rationale and specific applications were highlighted.
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