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Found: 26 174
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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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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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TMTP1-Modified Small Extracellular Vesicles Target BRAF Mutation in Anaplastic Thyroid Cancer Reversing Vemurafenib Resistance With CRISPR/Cas9 Delivery.
This study investigates a novel approach to overcome Vemurafenib resistance in BRAF-mutant Anaplastic thyroid carcinoma (ATC) using CRISPR/Cas9 gene editing and TMTP1-modified extracellular vesicles (TMTP1-sgBRAF-EVs). By knocking out the BRAF gene, the study elucidates Vemurafenib-induced ferroptosis mechanisms involving lipid peroxidation and reactive oxygen species (ROS) generation in ATC cells. The developed TMTP1-sgBRAF-EVs system demonstrates superior tumour-targeting and drug delivery capabilities, significantly enhancing Vemurafenib efficacy in both in vitro and in vivo models. This innovative combination of gene editing technology with a nanoparticle delivery system shows promising potential as a therapeutic strategy for treating aggressive BRAF-mutant ATC.
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Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional β-like cell generation.
Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in β-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation.
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Manipulation of Proteostasis Networks in Transgenic ZAAT Zebrafish via CRISPR-Cas9 Gene Editing.
The CRISPR-Cas9 genome editing system is used to induce mutations in genes of interest resulting in the loss of functional protein. A transgenic zebrafish α1-antitrypsin deficiency (AATD) model displays an unusual phenotype, in that it lacks the hepatic accumulation of the misfolding Z α1-antitrypsin (ZAAT) evident in human and mouse models. Here we describe the application of the CRISPR-Cas9 system to generate mutant zebrafish with defects in key proteostasis networks likely to be involved in the hepatic processing of ZAAT in this model. We describe the targeting of the atf6a and man1b1 genes as examples.
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How to create personalized gene editing platforms: Next steps toward interventional genetics.
How do we go from a single individual receiving a personalized gene-editing therapy to a future of "interventional genetics" in which such therapies are the standard of care? First and foremost: regulatory innovation.
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A Single Transcript CRISPR-Cas9 System for Multiplex Genome Editing in Plants.
The CRISPR-Cas9 system has been widely adopted in genome editing. By changing the 20 bp guide sequence, it can easily edit any sequence adjacent to a protospacer adjacent motif (PAM) in a genome. Multiplex genome editing could be accomplished with simultaneous expression of multiple single-guide RNAs (sgRNA). Given that sgRNAs are expressed by Pol III promoters, multiplex genome editing is conventionally done by assembly of multiple complete sgRNA expression cassettes together, which can be a challenge in vector construction. Here, we described a multiplex genome editing system based on a single transcript unit CRISPR-Cas9 (STU CRISPR-Cas9) expression system driven by a single Pol II promoter. It represents a novel approach for multiplex genome editing.
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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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RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing.
We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3'gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2 kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3'gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.
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Consumer acceptance of food crops developed by genome editing.
One of the major problems regarding consumer acceptance of genetically modified organisms (GMOs) is the possibility that their transgenes could have adverse effects on the environment and/or human health. Genome editing, represented by the CRISPR/Cas9 system, can efficiently achieve transgene-free gene modifications and is anticipated to generate a wide spectrum of plants. However, the public attitude against GMOs suggests that people will initially be unlikely to accept these plants. We herein explored the bottlenecks of consumer acceptance of transgene-free food crops developed by genome editing and made some recommendations. People should not pursue a zero-risk bias regarding such crops. Developers are encouraged to produce cultivars with a trait that would satisfy consumer needs. Moreover, they should carefully investigate off-target mutations in resultant plants and initially refrain from agricultural use of multiplex genome editing for better risk-benefit communication. The government must consider their regulatory status and establish appropriate regulations if necessary. The government also should foster communication between the public and developers. If people are informed of the benefits of genome editing-mediated plant breeding and trust in the relevant regulations, and if careful risk-benefit communication and sincere considerations for the right to know approach are guaranteed, then such transgene-free crops could gradually be integrated into society.
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