Targeting PARP1 in XRCC1-deficient sporadic invasive breast cancer or preinvasive ductal carcinoma in situ induces synthetic lethality and chemoprevention

© 2018 American Association for Cancer Research. Targeting PARP1 for synthetic lethality is a new strategy for breast cancers harboring germline mutations in BRCA. However, these mutations are rare, and reactivation of BRCA-mediated pathways may result in eventual resistance to PARP1 inhibitor therapy. Alternative synthetic lethality approaches targeting more common sporadic breast cancers and preinvasive ductal carcinoma in situ (DCIS) are desirable. Here we show that downregulation of XRCC1, which interacts with PARP1 and coordinates base excision repair, is an early event in human breast cancer pathogenesis. XRCC1-deficient DCIS were aggressive and associated with increased risk of local recurrence. Human invasive breast cancers deficient in XRCC1 and expressing high PARP1 levels also manifested aggressive features and poor outcome. The PARP1 inhibitor olaparib was synthetically lethal in XRCC1-deficient DCIS and invasive breast cancer cells. We conclude that targeting PARP1 is an attractive strategy for synthetic lethality and chemoprevention in XRCC1-deficient breast cancers, including preinvasive DCIS. Significance: These findings show that loss of XRCC1, which is associated with more malignant DCIS, can be exploited by PARP inhibition, suggesting its application as a promising therapeutic and chemoprevention strategy in XRCC1-deficient tumor cells

Utilizing CRISPR/Cas9 Gene Editing Determine the Role of cnr1 during Zebrafish Development

In a world where cannabinoids are being used more frequently as for medicinal purposes, the importance for research on cannabinoid biology is steadily increasing. The goal of this study was to gain a greater understanding of the effect cannabinoids have on developing zebrafish embryos. Work in this area of developmental biology could give way to an understanding of the safety of a human fetus during pregnancy with a mother who uses cannabis medicinal therapy. The CRISPR/Cas 9 system was utilized in this study to genetically edit the zebrafish embryo genome to remove the function of the Cannabinoid Receptor 1 (cnr1). Cnr1 is a one of the receptors that is a part of the endocannabinoid system, the other being cnr2, which serve as receptors for cannabinoid compounds and endogenous Anandamide. CRISPR/ cas 9 is an effective tool used to mutate genes in a way that allow for loss of function studies, which can provide information beyond the effects that occur in embryos that still have crn1 fully intact. With this in mind, our research was designed to observe behavioral differences in young cnr1 mutant fish and fish who have receptor when exposed to cannabinoids. This research could provide an opportunity to study the toxicology of mutant embryos when exposed to cannabinoid agonists and help determine which receptor they are targeting. This research is clinically important for future study and understanding the effects of cannabinoid therapy on early developmental patterning

Scientific Communication and CRISPR

Genome editing technologies have the capability of providing a quick, relatively inexpensive method of plant breeding that could greatly expand breeding efforts across species. CRISPR-Cas9 has become one of the leading forms of genome editing, but the level of investment and number of products developed with the technology will be greatly affected by both regulatory decisions made around the world and the level of consumer acceptance. The ability of scientists who are involved with genome editing research to communicate with non-experts will impact the outcomes of both factors. This paper reviews available literature on scientific communication, provides a high-level discussion on the CRISPR-Cas9 system of genome editing, and links the two topics with a discussion of how scientific communication will affect the utilization of CRISPR-Cas9, providing recommendations for those who wish to engage in the discussion. This review of the literature makes it apparent that it will not be sufficient to solely educate the public about gene editing technologies; rather, a dialogue must be opened that will both educate and address regulatory and consumer concerns in an honest, transparent way

Apoptosis in Crithidia fasciculata

Crithidia fasciculata belongs to a group of parasites called kinetoplastids that comprise many important human pathogens. Evidence of apoptosis has been found in these parasites with pathways that appear to be different than in mammalian cells. Therefore, careful characterization of these pathways can provide ways to manipulate parasite infection which could be used to create better treatments for these diseases. In this study, potential apoptosis genes conserved across all kinetoplastid parasites were identified using gene prediction programs in Tri-TrypDB and BLAST searches. Homologous genes were identified in C. fasciculata and a comprehensive q-PCR analysis showed differential upregulation upon induction of apoptosis. One of the genes significantly changed was Bax1 inhibitory gene (Bax1i), an inhibitor of the putative apoptosis promoting Bax1. In order to characterize this gene further we made gene modification constructs for tagging and gene deletion using the CRISPR-Cas-9 system. A homologous repair template was created for Bax1i using 500 bp homology arms and a drug resistance gene using a fusion PCR protocol. Constructs were made using both Puromycin and Blasticidin resistance genes. We have successfully created and optimized the fusion PCR protocol for generation of 3.5Kb drug repair cassettes. The same process was repeated for Phosphoglycerate mutase family member 5 (PGAM5). Drug selection trials using Puromycin found the optimum concentration of drug is 50 µg/mL. Blasticidin trials are still being performed. The optimization of the fusion PCR protocol and drug selection procedure, along with the identification of genes done in this project will be important for continuing work

CRISPR/Cas9-Mediated Genome Editing of Y705 Residue of STAT3 for Genetic Validation of Small Molecule Inhibitors In Breast Cancer Cells​

https://openworks.mdanderson.org/sumexp23/1122/thumbnail.jp

Utilizing CRISPR/Cas9 Gene Editing Determine the Role of cnr1 during Zebrafish Development

The CRISPR-Cas 9 system is a process used to create mutations, insertions, or deletions within a desired gene. It can be used to create mutant organisms necessary to study the function and impact of a particular gene1. Cannabinoids are active chemicals found in cannabis, the most common of which being cannabinol (CBD)2. Cannabinoid receptors are an integral part of the endocannabinoid system, recognizing cannabinoid signals to promote physiological processes like pain sensation, memory, mood, and appetite3. Studying these cannabinoid receptors can provide insight into how cannabis can affect bodily processes. Zebrafish are small translucent freshwater fish residing the minnow family, allowing for developing structures to be observed very easily. This optical transparency paired with the inexpensiveness and quick reproduction of zebrafish makes them a very good model system for research. Zebrafish share 70% similarity in their genome with humans. The zebrafish genome includes two cannabinoid receptor genes- cannabinoid receptor 1 (cnr1) and cannabinoid receptor 2 (cnr2)5. cnr1 has been shown to play an important role in regulating aspects of brain function, including mood, anxiety, appetite, and memory, and locomotor activity within zebrafish5. The purpose of this study was to determine the developmental effects of cnr1 within embryos of zebrafish, followed by toxicological and behavioral observations performed after their maturation to further this cause. The data obtained from this model could provide insight into the effects observed on human fetal development within a mother using cannabis medicinally; which is not completely understood. Citations 1. Damm, Erich, Glenn, Nicole. CRISPR-Cas9 Protocol. Belmont University, 22 Nov. 2019. 2. “Cannabinoids.” Learn About Marijuana: Factsheets: Cannabinoids, National Cannabis Prevention and Information Centre, adai.uw.edu/marijuana/factsheets/cannabinoids.htm 3. Luchtenburg, F.J., Schaaf, M.J.M. & Richardson, M.K. Functional characterization of the cannabinoid receptors 1 and 2 in zebrafish larvae using behavioral analysis. Psychopharmacology 236, 2049–2058 (2019). https://doi.org/10.1007/s00213-019-05193-4 4. Burke, Elizabeth. “Why Use Zebrafish to Study Human Diseases?” National Institutes of Health, U.S. Department of Health and Human Services, 14 Oct. 2016, irp.nih.gov/blog/post/2016/08/why-use-zebrafish-to-study-human-diseases. 5. Krug, Randall & Clark, Karl. (2015). Elucidating Cannabinoid Biology in Zebrafish (Danio rerio). Gene. 570. 10.1016/j.gene.2015.07.036

Analysis of gene function by CRISPR/Cas9 deletions and transcriptomics

[SPA] CRISPR-Cas9, una técnica revolucionaria actualmente utilizada para modificar el genoma de una manera precisa, se basa en los mecanismos de reparación del ADN activados localmente por la rotura del ADN. CRISPR se asocia con una endonucleasa llamado Cas9. Este complejo está dirigido por un gRNA (guía) que confiere especificidad de acción para una secuencia de ADN dada. El objetivo del presente trabajo es utilizar la técnica CRISPR / Cas9 para desarrollar una serie de plantas mutagenéticos utilizando la transformación in vitro. [ENG] CRISPR-Cas9, a revolutionary technique currently used to modify the genome in a precise way, is based on DNA repair mechanisms activated locally by DNA breakage. CRISPR is associated with an endonuclease called Cas9. This complex is directed by a gRNA (guide) conferring specificity of action to a given DNA sequence. The aim of the present work is to use the CRISPR / Cas9 technique to develop a series of mutagenetic plants using in vitro transformation.The experiments will be conducted at the Institute of Plant Biotechnology (IBV), Cartagena, Polytechnical University of Cartagena (UPCT

Characterization of Toxoplasma gondii Calcium regulator proteins TGGT1_253640 and TGGT1_222060 for Toxoplasma gondii growth and invasion

Toxoplasma gondii is an opportunistic apicomplexan parasite infecting humans and livestock. Infection in immunocompromised individuals can cause neurological damage and infection during pregnancy can lead to fetal death. These parasites engage in a complex life cycle, involving repeated invasion of the host cell and egress from the host cell. Calcium signaling is an important regulatory mechanism for many essential processes in the parasite, including gliding motility (actomyosin-dependent mode of motion), invasion, and egress. Our work here focuses on two previously uncharacterized calcium regulator proteins (TGGT1_253640 and TGGT1_222060). To characterize the role of these proteins in parasite viability and calcium regulation, we fused an auxin degron system using CRISPR-cas9 gene modification technique. We have localized the TGGT1_253640 to endoplasmic reticulum of the parasite. The protein is downregulated upon the addition of auxin as early as four hours thus providing a tool to study the function of the protein. Future work aims to determine the role of these proteins in parasite growth and fitness using growth and replication assays

Targeting FGF8a Promoter for Gene Expression

In this paper, I will be assisting in developing a new method of characterizing communication between the enhancers and promoter within the fgf8a gene in vertebrates, specifically in zebrafish. The fgf8a gene controls important growth functions and regulates developmental processes in zebrafish. Currently, there is no understanding on how the enhancers function within the fgf8a gene, thus my research will further describe gene regulation and the role of these enhancers. My expected outcomes are to create specific engineered CRISPR RNA that will target the gene promoter and to potentially visualize the gene promoter within the nucleus. Previous research studied the role of enhancers in deceased samples and our research will develop an approach for imaging the promoter portion of the gene in real-time. Targeting the promoter is the first step to identifying the interaction between the promoter and enhancers in the fgf8a gene. Two CRISPR RNA sequences developed to target the fgf8a promoter were inserted into zebrafish embryos to test against a control. We conducted an inexpensive, rapid genomic extraction method with a high-resolution melt assay that is more sensitive and allows early detection of CRISPR-induced indels in zebrafish. The results showed that the CRISPR RNA sequences caused a mutation in genetic expression and confirmed targeting of the fgf8a promoter. These findings will lead us to a step closer in visualizing the interaction of the enhancers and promoters of the gene to further understand its regulation. Demonstrating and defining how this gene expresses itself in zebrafish can advance our understanding of gene expression in human development