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CRISPR/Cas9, a promising approach for the treatment of β-thalassemia: a systematic review.
The CRISPR/Cas9 technique is easily programmable, fast, more powerful, and efficient at generating a mutation compared to previous gene therapy methods. β-thalassemia is the most common autosomal recessive disorder worldwide. Appropriate genomic changes in the β gene can be modified to alleviate the symptoms of the disease using the CRISPR/Cas9 system. PubMed/Medline, Scopus, Web of Science, and SID databases were searched in Persian and English from February 2000 to September 2022. Finally, 39 articles had inclusion criteria which were reviewed by two separate individuals. Among the reviewed articles, articles were divided into three categories. In the first group, studies attemped to increase the expression of γ-globin and production of hemoglobin F. The strategy of second group of studies were the reduction of the α-globin chain to prevent hemolysis of RBCs by accumulation of excessive α-globins. The third group corrected the mutations causing β-thalassemia. Studies have shown that the genome of β-thalassemia patients can be modified using the CRISPR/Cas9 technique, and this approach might be promising for the treatment of β-thalassemia.
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The Cas9 Hammer-and Sickle: A Challenge for Genome Editors.
Genome editing using CRISPR-Cas9 has produced a functional cure for a small number of patients with sickle cell disease and beta-thalassemia. Rather than repairing the causative mutation, this striking outcome was attained by the knockout of a lineage-specific regulatory element for a gene, BCL11A, that controls fetal hemoglobin levels: a first example of clinical success in targeting a locus initially identified in a genome-wide association study, and formal proof of the "in the age of CRISPR, the entire genome is a druggable target" notion. This remarkable development, along with advancement to the clinic of several additional editing-based approaches to the hemoglobinopathies, highlights a sense of urgency in accelerating scientific, regulatory, and public health innovation that will allow broad and equitable access to editing-based cures.
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CRISPR Genome Editing and the Study of Chagas Disease.
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is an illness that affects 6-8 million people worldwide and is responsible for approximately 50,000 deaths per year. Despite intense research efforts on Chagas disease and its causative agent, there is still a lack of effective treatments or strategies for disease control. Although significant progress has been made toward the elucidation of molecular mechanisms involved in host-parasite interactions, particularly immune evasion mechanisms, a deeper understanding of these processes has been hindered by a lack of efficient genetic manipulation protocols. One major challenge is the fact that several parasite virulence factors are encoded by multigene families, which constitute a distinctive feature of the T. cruzi genome. The recent advent of the CRISPR/Cas9 technology represented an enormous breakthrough in the studies involving T. cruzi genetic manipulation compared to previous protocols that are poorly efficient and required a long generation time to develop parasite mutants. Since the first publication of CRISPR gene editing in T. cruzi, in 2014, different groups have used distinct protocols to generated knockout mutants, parasites overexpressing a protein or expressing proteins with sequence tags inserted in the endogenous gene. Importantly, CRISPR gene editing allowed generation of parasite mutants with gene disruption in multi-copy gene families. We described four main strategies used to edit the T. cruzi genome and summarized a large list of studies performed by different groups in the past 7 years that are addressing several mechanisms involved with parasite proliferation, differentiation, and survival strategies within its different hosts.
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Enhancing Animals is "Still Genetics": Perspectives of Genome Scientists and Policymakers on Animal and Human Enhancement.
BACKGROUND: Nonhuman animals are regularly enhanced genomically with CRISPR and other gene editing tools as scientists aim at better models for biomedical research, more tractable agricultural animals, or animals that are otherwise well suited to a defined purpose. This study investigated how genome editors and policymakers perceived ethical or policy benefits and drawbacks for animal enhancement and how perceived benefits and drawbacks are alike, or differ from, those for human genome editing. METHODS: We identified scientists through relevant literature searches as well as conference presentations. Policymakers were identified through rosters of genome editing oversight groups (e.g., International Commission on the Clinical Use of Human Germline Genome Editing, World Health Organization) or efforts aimed at influencing policy (e.g., deliberative democracy groups). Interviews covered participants' views on ethical differences between interventions affecting somatic or germline cells and distinctions between using gene editing for disease treatment, prevention, and enhancement purposes. RESULTS: Of the 92 participants interviewed, 81 were genome editing scientists, and 33 were policymakers, with 22 interviewees being both scientists and policymakers. Multiple areas were identified in which the ethical implications of genomic enhancements for nonhuman animals differ from those for human animals including with respect to experiential welfare; germline edits; environmental sustainability; and justice. CONCLUSIONS: Overall, respondents viewed that animal enhancement is unburdened by the ethical complexities of human enhancement. These views may be related to participant perceptions of animals' lesser moral status and because germline editing in animals is common practice.
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Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.
We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar 'Madness Midnight' has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T0 plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer's field.
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Creating cell lines for mimicking diseases.
Cell lines can be good models for the disease they are derived from but can also be used to study general physiological and pathological processes. They can also be used to generate cell models of diseases when primary cultures are not available. Recent genome editing tools have been very promising tools toward creating cell models to mimic diseases in vitro. In this chapter, we highlight techniques used to obtain genome-edited cell lines, including cell line selection, transfection and gene editing tools available, together with methods of phenotype characterization and, lastly, a few examples of how in vitro disease models were created using CRISPR-Cas9.
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Multiplexed CRISPR-Cas9 mutagenesis of rice PSBS1 noncoding sequences for transgene-free overexpression.
Understanding CRISPR-Cas9's capacity to produce native overexpression (OX) alleles would accelerate agronomic gains achievable by gene editing. To generate OX alleles with increased RNA and protein abundance, we leveraged multiplexed CRISPR-Cas9 mutagenesis of noncoding sequences upstream of the rice PSBS1 gene. We isolated 120 gene-edited alleles with varying non-photochemical quenching (NPQ) capacity in vivo-from knockout to overexpression-using a high-throughput screening pipeline. Overexpression increased OsPsbS1 protein abundance two- to threefold, matching fold changes obtained by transgenesis. Increased PsbS protein abundance enhanced NPQ capacity and water-use efficiency. Across our resolved genetic variation, we identify the role of 5'UTR indels and inversions in driving knockout/knockdown and overexpression phenotypes, respectively. Complex structural variants, such as the 252-kb duplication/inversion generated here, evidence the potential of CRISPR-Cas9 to facilitate significant genomic changes with negligible off-target transcriptomic perturbations. Our results may inform future gene-editing strategies for hypermorphic alleles and have advanced the pursuit of gene-edited, non-transgenic rice plants with accelerated relaxation of photoprotection.
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Functionally Tunable Star-Shaped Multivalent crRNAs for Photocontrol CRISPR/Cas Editing.
Clustered regularly interspaced shortpalindromic repeats/CRISPR-associated (CRISPR/Cas)-based genome editing has significantly advanced genetic engineering due to its precision, simplicity, and versatility. However, achieving precise spatial and temporal control remains challenging, restricting therapeutic and research applications. Herein, we introduce a novel class of star-shaped, multivalent crRNAs engineered for precise spatiotemporal control of CRISPR/Cas9 and Cas12a editing systems. These crRNAs are synthesized via single-site chemical modification and can be efficiently purified. By integrating distinct photo-responsive chemical linkages, we achieved selective activation of crRNA activity upon irradiation with specific wavelengths, enabling orthogonal regulation of multiple genetic targets simultaneously. This method demonstrated robust OFF-ON switching capabilities in vitro, characterized by minimal leakage and rapid activation. Importantly, the approach also proved highly effective for temporally controlled gene editing in mammalian cells in vivo, achieving considerable editing efficiency following brief photoactivation. Due to its target sequence-independent, single-site modification design, this strategy may serve as a universal solution for diverse CRISPR/Cas systems, eliminating cumbersome optimization processes. Future advancements incorporating long-wavelength responsive and reversible linkers promise further enhancement of tissue penetration and control, significantly broadening the applicability and impact of this approach in biological research and therapeutic interventions.
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Insights into Prime Editing Technology: A Deep Dive into Fundamentals, Potentials, and Challenges.
As the most versatile and precise gene editing technology, prime editing (PE) can establish a durable cure for most human genetic disorders. Several generations of PE have been developed based on an editor machine or prime editing guide RNA (pegRNA) to achieve any kind of genetic correction. However, due to the early stage of development, PE complex elements need to be optimized for more efficient editing. Smart optimization of editor proteins as well as pegRNA has been contemplated by many researchers, but the universal PE machine's current shortcomings remain to be solved. The modification of PE elements, fine-tuning of the host genes, manipulation of epigenetics, and blockage of immune responses could be used to reach more efficient PE. Moreover, the host factors involved in the PE process, such as repair and innate immune system genes, have not been determined, and PE cell context dependency is still poorly understood. Regarding the large size of the PE elements, delivery is a significant challenge and the development of a universal viral or nonviral platform is still far from complete. PE versions with shortened variants of reverse transcriptase are still too large to fit in common viral vectors. Overall, PE faces challenges in optimization for efficiency, high context dependency during the cell cycling, and delivery due to the large size of elements. In addition, immune responses, unpredictability of outcomes, and off-target effects further limit its application, making it essential to address these issues for broader use in nonpersonalized gene editing. Besides, due to the limited number of suitable animal models and computational modeling, the prediction of the PE process remains challenging. In this review, the fundamentals of PE, including generations, potential, optimization, delivery, in vivo barriers, and the future landscape of the technology are discussed.
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DGK and DZHK position paper on genome editing: basic science applications and future perspective.
For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.
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