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Gene editing for sickle cell disease and transfusion dependent thalassemias- A cure within reach.
Sickle cell disease (SCD) is associated with significant morbidity and shortened life expectancy. Similarly, patients with transfusion dependent beta thalassemia (TdT) require life-long transfusion therapy, chelation therapy and significant organ dysfunction. Allogeneic transplantation from a matched family donor provided the only curative option for patients with SCD and TdT. Unfortunately, less than 20% of patients have a fully matched related donor and results using unrelated donor transplant were associated with high rate of complications. Ex vivo gene therapy through globin gene addition has been investigated extensively and recent encouraging preliminary data resulted in regulatory approval in patients with TdT. Recent improvements in our understanding of the molecular pathways controlling erythropoiesis and globin switching from fetal hemoglobin to adult hemoglobin offer a new and exciting therapeutic options. Rapid and substantial advances in genome editing tools using CRISPR/Cas9, have raised the possibility of genetic editing and correction in patient derived hematopoietic stem and progenitor cells. We will review results of gene editing approach that can induce fetal hemoglobin production in patients with SCD and TdT.
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Single-Molecule Imaging of Endogenous Proteins.
Single-molecule imaging is a technique of choice to investigate the dynamic nanoscale organization of proteins of interest, revealing a wealth of new information on how proteins perform their biological function. However, overexpression, which is often used in this context, can alter biological functions. It is therefore useful to implement tools to visualize and track endogenous proteins in living cells and observe their dynamic clustering and motion within their native environment. Herein, we describe two approaches: Fluorescent intrabody Localization Microscopy (FiLM) and gene-editing (CRISPR/Cas9) and provide a workflow including the design of plasmid backbones used to track endogenous proteins by either i) introducing nanobodies that target endogenous proteins to perform single-particle tracking or ii) gene-editing cells in culture to express tagged endogenous proteins at biologically relevant expression levels.
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Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair.
Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf. Our experiments revealed that gene editing could effectively restore Fancf function via error-prone end joining resulting in a 27% increased survival in the presence of mitomycin C. In addition, templated gene correction could be achieved after double strand or single strand break formation. Although templated gene editing efficiencies were low (≤6%), FA corrected embryonic stem cells acquired a strong proliferative advantage over non-corrected cells, even without imposing genotoxic stress. Notably, Cas9 nickase activity resulted in mono-allelic gene editing and avoidance of undesired mutagenesis. In conclusion: DNA repair defects associated with FANCF deficiency do not prohibit CRISPR/Cas9 gene correction. Our data provide a solid basis for the application of pre-clinical models to further explore the potential of gene editing against FA, with the eventual aim to obtain therapeutic strategies against bone marrow failure.
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Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7.
This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.
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The Potential Therapeutic Applications of CRISPR/Cas9 in Colorectal Cancer.
The application of the CRISPR-associated nuclease 9 (Cas9) system in tumor studies has led to the discovery of several new treatment strategies for colorectal cancer (CRC), including the recognition of novel target genes, the construction of animal mass models, and the identification of genes related to chemotherapy resistance. CRISPR/Cas9 can be applied to genome therapy for CRC, particularly regarding molecular-targeted medicines and suppressors. This review summarizes some aspects of using CRISPR/- Cas9 in treating CRC. Further in-depth and systematic research is required to fully realize the potential of CRISPR/Cas9 in CRC treatment and integrate it into clinical practice.
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A method for generating genome edited plant lines from CRISPR-transformed Shanxin poplar plants.
Due to the reason of low efficiency of mutation in CRISPR-editing, a high frequency of CRISPR transformed plant lines failing in mutation had been generated and had to be discarded. In the present study, we built a method to increase the efficiency of CRISPR-editing. We used Shanxin poplar (Populus davidiana×P. bolleana) as the study material, and CRISPR-editing system was first built to generate the CRISPR-transformed lines. The line that failed in CRISPR-editing was used for improving the efficiency of mutation, which was treated with heat (37 °C) to improve the cleaving activity of Cas9, leading to increased frequency of the cleaved DNA. Our results indicated that 87-100% of cells in CRISPR-transformed plants whose DNA had been cleaved by heat treatment, and the heat treatment plants were then cut into explants to differentiate adventitious buds. Each differentiated bud can be considered as an independent line. Twenty independent lines were randomly selected for analysis, and all of them had been mutated by CRISPR editing, displaying 4 types of mutation. Our results indicated that heat treatment combined with re-differentiation can generate CRISPR-edited plants efficiently. This method could conquer the problem of low mutation efficiency of CRISPR-editing in Shanxin poplar, and will have a wide application in plant CRISPR-editing.
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Macrophage-Specific in Vivo Gene Editing Using Cationic Lipid-Assisted Polymeric Nanoparticles.
The CRISPR/Cas9 gene editing technology holds promise for the treatment of multiple diseases. However, the inability to perform specific gene editing in targeted tissues and cells, which may cause off-target effects, is one of the critical bottlenecks for therapeutic application of CRISPR/Cas9. Herein, macrophage-specific promoter-driven Cas9 expression plasmids (pM458 and pM330) were constructed and encapsulated in cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN). The obtained nanoparticles encapsulating the CRISPR/Cas9 plasmids were able to specifically express Cas9 in macrophages as well as their precursor monocytes both in vitro and in vivo. More importantly, after further encoding a guide RNA targeting Ntn1 (sgNtn1) into the plasmid, the resultant CLANpM330/sgNtn1 successfully disrupted the Ntn1 gene in macrophages and their precursor monocytes in vivo, which reduced expression of netrin-1 (encoded by Ntn1) and subsequently improved type 2 diabetes (T2D) symptoms. Meanwhile, the Ntn1 gene was not disrupted in other cells due to specific expression of Cas9 by the CD68 promoter. This strategy provides alternative avenues for specific in vivo gene editing with the CRISPR/Cas9 system.
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[CRISPR/Cas9 and gene therapy].
The development of techniques that allow the precise and efficient edition of the genome of living cells is one of the main goals of biomedical research. Over the last few decades, a number of genome editing tools have been developed, the most prominent being the CRISPR/Cas9 system, a bacterial defense mechanism that has been redesigned for its use in other cellular systems. The accessibility, both technical and economical, and the enormous potential of CRISPR/Cas9 have contributed to an almost unprecedented revolution in the biomedical sciences and represent an important step forward in the field of gene therapy that needs, however, to be taken cautiously.
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Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi.
The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have generated homogeneous populations of genetically modified parasites in 4-5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the gene. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines generated by CRISPR/Cas9, and C-terminal tagging of endogenous genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.
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Considerations on genetic engineering: regarding the birth of twins subjected to gene edition.
In this essay, the bioethical implications of the recent genetic manipulation in human embryos with CRISPR-Cas9 to eliminate the CCR5 gene and the birth of a pair of discordant twin girls are analyzed. The experiment was disseminated via social media. The main bioethical flaws identified include the justification of the model, the informed consent process and the lack of disclosure of evident conflicts of interest. The consequences of the experiment on the life of the twins that were born were not properly evaluated, such as the impact on their autonomy, the alleged benefits to be received and the future risks of harm during their lifetime. Having manipulated the germ cell line, the effects on their future offspring were not considered. This type of actions negatively affects the way society conceives science. Genetic engineering should be reserved to the basic experimental context or as clinical research for the correction of known serious diseases of genetic origin under strict regulatory and bioethical supervision and using a gradualist approach in accordance with the advances of gene editing techniques.
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