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Delivery and Specificity of CRISPR-Cas9 Genome Editing Technologies for Human Gene Therapy.
Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) technology is revolutionizing the study of gene function and likely will give rise to an entire new class of therapeutics for a wide range of diseases. Achieving this goal requires not only characterization of the technology for efficacy and specificity but also optimization of its delivery to the target cells for each disease indication. In this review we survey the various methods by which the CRISPR-Cas9 components have been delivered to cells and highlight some of the more clinically relevant approaches. Additionally, we discuss the methods available for assessing the specificity of Cas9 editing; an important safety consideration for development of the technology.
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The Arabidopsis R-SNARE protein YKT61 is essential for gametophyte development.
Gametophyte development is a pre-requisite for plant reproduction and seed yield; therefore, studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture. The biogenesis and dynamics of endomembrane compartments are critical for cell survival, and their regulatory mechanisms are just beginning to be revealed. Here, we report that the Arabidopsis thaliana SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein YKT61 is essential for both male and female gametogenesis. By using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing, we demonstrated that male and female gametophytes carrying YKT61 loss-of-function alleles do not survive. Specifically, loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes. A three-base-pair deletion in YKT61 in the ykt61-3 mutant resulted in a single-amino acid deletion in the longin domain of YKT61; the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association, suggesting that the N-terminal longin domain of YKT61 plays multiple roles in its function. This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9-mediated editing and expression from a Cas9-resistant transgene.
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Genome Editing in Zebrafish Using CRISPR-Cas9: Applications for Developmental Toxicology.
Environment-gene interactions have a powerful impact on embryo development. The ability to precisely edit the genome makes it possible to address questions concerning the specific roles that genes or variants play in modulating the response to environmental challenges. In this chapter, we provide a simplified protocol using CRISPR-Cas9 ribonucleoproteins for genome editing in the zebrafish model organism. The genetic manipulation can then be coupled with chemical screens to identify and understand the mechanism behind toxicants or compounds that modulate development.
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Precision pest management: Genome editing tools, specifically CRISPR/Cas9 and future prospects.
The growing resistance to synthetic insecticides and Bt toxins, alongside persistent crop losses despite heavy pesticide application, highlights the urgent need for safer, sustainable and efficient pest management strategies. This review presents genome editing as a precise and versatile approach to reduce pest impact by altering fertility, feeding patterns or vulnerability, while protecting beneficial organisms. Among the genome editing tools, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) is one of the most promising genome editing techniques in insects. It facilitates targeted functional studies, integration with RNAi and dual-expression systems and gene drive applications. Deployment is envisioned in two phases, initial laboratory modification followed by regulated field release, with a strong emphasis on biosafety through terminator genes, marked individuals for gene flow monitoring, optimized dosages, stringent screening and long-term ecological surveillance, along with transparency and adherence to international safety protocols. Significant challenges encompass delivery efficiency, identification of edits, off-target mutations, dose-related efficacy and sterility, unstable transmission and resistance development. Innovations such as base and prime editing minimize unintended mutations by circumventing double-stranded breaks (DSBs), while paratransgenic strategies targeting gut symbionts offer supplementary avenues; plant-mediated insect gene editing emerges as a promising frontier. Overall, carefully regulated trials aligned with policy frameworks and stakeholder involvement are vital to assess effectiveness in natural environments and achieve targeted, dependable and ecologically responsible pest control.
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A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish.
Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish.
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Combination of CRISPR/Cas9 System and CAR-T Cell Therapy: A New Era for Refractory and Relapsed Hematological Malignancies.
Chimeric antigen receptor T (CAR-T) cell therapy is the novel treatment strategy for hematological malignancies such as acute lymphoblastic leukemia (ALL), lymphoma and multiple myeloma. However, treatment-related toxicities such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) have become significant hurdles to CAR-T treatment. Multiple strategies were established to alter the CAR structure on the genomic level to improve efficacy and reduce toxicities. Recently, the innovative gene-editing technology-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease9 (Cas9) system, which particularly exhibits preponderance in knock-in and knockout at specific sites, is widely utilized to manufacture CAR-T products. The application of CRISPR/Cas9 to CAR-T cell therapy has shown promising clinical results with minimal toxicity. In this review, we summarized the past achievements of CRISPR/Cas9 in CAR-T therapy and focused on the potential CAR-T targets.
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Is Pooled CRISPR-Screening the Dawn of a New Era for Functional Genomics.
Functional genomics aims to develop an in-depth understanding of how specific gene dysfunctions are related to diseases. A common method for investigating the genome and its complex functions is via perturbation of the interactions between the DNA, RNA and their protein respective protein derivatives. Commonly, arrayed and pooled genetic screens are utilized to achieve this and in recent years have been fundamental in achieving the current level of understanding for gene dysfunctions. However, they are limited in specific aspects which scientists have attempted to address. Clustered regularly palindromic repeats (CRISPR)-based methods for genetic screens have in recent years become more prevalent but crucially shared similar properties to previous methods and failing to provide a distinct advantage over previous methods. CROP-seq, Perturb-seq, and CRISPR-seq have combined CRISPR and single-cell RNA-sequencing (scRNA-seq) and is the newest addition to the geneticist's arsenal, providing scientists with methods to edit DNA with improved speed, accuracy, and efficiency which could usher us into a new era of study methods for functional genomics. We briefly overview the CRISPR-Cas9 systems, the evolution of genetic screening in recent years, and evaluate and discuss the significance of CROP-seq, Perturb-seq, and CRISPR-seq.
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CRISPR-Mediated Gene Editing: Scientific and Ethical Issues.
There remains substantial evidence to warrant great concern over the poor efficiency and specificity of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genetic modification (GM), despite relatively minor improvements compared to other GM methods. These issues cause persistent, adverse, ethical, and scientific consequences for GM animals, which may never be sufficiently resolvable.
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Potentials of CRISPR in liver research and therapy.
The CRISPR technology is rapidly transforming the field of liver research by its versatility and easy use. In vivo gene editing of hepatocytes in adult mice can be achieved using a broad toolbox for both fundamental research and development of therapeutic strategies for future clinical applications. Recent studies showed that CRISPR has a real potential to treat hereditary liver diseases as well as virally induced pathologies. This short review recapitulates very recent advancements regarding the use of CRISPR in liver research and therapy.
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Folate Receptor-Mediated Delivery of Cas9 RNP for Enhanced Immune Checkpoint Disruption in Cancer Cells.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system offers great opportunities for the treatment of numerous diseases by precise modification of the genome. The functional unit of the system is represented by Cas9/sgRNA ribonucleoproteins (RNP), which mediate sequence-specific cleavage of DNA. For therapeutic applications, efficient and cell-specific transport into target cells is essential. Here, Cas9 RNP nanocarriers are described, which are based on lipid-modified oligoamino amides and folic acid (FolA)-PEG to realize receptor-mediated uptake and gene editing in cancer cells. In vitro studies confirm strongly enhanced potency of receptor-mediated delivery, and the nanocarriers enable efficient knockout of GFP and two immune checkpoint genes, PD-L1 and PVR, at low nanomolar concentrations. Compared with non-targeted nanoparticles, FolA-modified nanocarriers achieve substantially higher gene editing including dual PD-L1/PVR gene disruption after injection into CT26 tumors in vivo. In the syngeneic mouse model, dual disruption of PD-L1 and PVR leads to CD8+ T cell recruitment and distinct CT26 tumor growth inhibition, clearly superior to the individual knockouts alone. The reported Cas9 RNP nanocarriers represent a versatile platform for potent and receptor-specific gene editing. In addition, the study demonstrates a promising strategy for cancer immunotherapy by permanent and combined immune checkpoint disruption.
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