Therapeutic applications of CRISPR/Cas9 in breast cancer and delivery potential of gold nanomaterials.

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites.

Due to their superiority in the simple design and precise targeting, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have attracted significant interest for biosensing. On the one hand, CRISPR-Cas systems have the capacity to precisely recognize and cleave specific DNA and RNA sequences. On the other hand, CRISPR-Cas systems such as orthologs of Cas9, Cas12, and Cas13 exhibit cis-cleavage or trans-cleavage activities after recognizing the target sequence. Owing to the cleavage activities, CRISPR-Cas systems can be designed for biosensing by degrading tagged nucleic acids to produce detectable signals. To meet the requirements of point-of-care detection and versatile signal readouts, gold nanomaterials with excellent properties such as high extinction coefficients, easy surface functionalization, and biocompatibility are implemented in CRISPR-Cas-based biosensors. In combination with gold nanomaterials such as gold nanoparticles, gold nanorods, and gold nanostars, great efforts are devoted to fabricating CRISPR-Cas-based biosensors for the detection of diverse targets. This review focuses on the current advances in gold nanomaterials-implemented CRISPR-Cas-based biosensors, particularly the working mechanism and the performance of these biosensors. CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13a are discussed and highlighted. Meanwhile, prospects and challenges are also discussed in the design of biosensing strategies based on gold nanomaterials and CRISPR-Cas systems.

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

e18500 Background: Approximately 1.4 million adult Americans identify as transgender (TG) or non-binary (NB), a number that has steadily increased over time. In this population, cancer risk is unclear, and screening and treatment guidelines are lacking. We sought to assess TG and NB persons’ perceptions towards breast and cervical cancer screening, risk of cancer development, and thoughts towards gender-affirming hormone therapy in the setting of a hormone-receptor positive breast cancer. Methods: A single-institution online survey was administered from October 2021-January 2022 at our comprehensive LGBTQ+ Inclusion Health Clinic. Participants with female sex at birth were asked about breast and cervical cancer, while those assigned male sex at birth were exclusively asked about breast cancer. A 5-point Likert scale was used to assess attitudes toward cancer screening and concerns regarding cancer development. Results: 40 patient responses were collected: 13% were TG women, 45% TG men, 23% NB, and 20% identified as other (i.e., agender, genderqueer, etc). 71% were assigned female sex at birth (59% of whom had chest masculinization surgery), 27% were assigned male sex at birth, and one individual was intersex at birth. 52.5% were age < 30, 84% were Caucasian, 65% had at least a bachelor’s degree, and all but one respondent had health insurance. The majority reported they were not familiar with breast (77%) or cervical (60%) cancer screening recommendations for their sex-assigned at birth or current gender identity. 23% reported concern regarding breast cancer development and cited family history as the primary reason. In patients age > 40, 50% had a mammogram (MMG) in the past 10 years. When presented with information regarding screening MMG and automated breast ultrasonography (ABUS), 78% reported they would prefer ABUS over MMG for breast cancer screening. 84% of respondents were currently using or had previously used gender-affirming hormone therapy, and of these, 61% reported they would stop therapy in the event they developed a hormone-receptor positive breast cancer. In patients assigned female sex at birth, 25% had a hysterectomy and, in those who still had a cervix, 50% reported having a Pap smear in the past 5 years and 38% were concerned about cervical cancer development. Conclusions: This survey identified that > 60% of TG and NB individuals are unaware of breast and cervical cancer screening guidelines, > 20% are concerned about breast and cervical cancer development, and < 50% of patients eligible for breast and cervical cancer screening had undergone screening in recent years. Additionally, in the setting of a hormone-receptor positive breast cancer, only 61% would consider stopping gender-affirming hormone therapy. Further data on the risk of breast and cervical cancer development and incidence in TG and NB persons is needed to inform optimal screening and treatment guidelines.

Breast cancer (BC) is the second most commonly diagnosed cancer in the world. BC develops due to dysregulation of transcriptional profiles, substantial interpatient variations, genetic mutations, and dysregulation of signaling pathways in breast cells. These events are regulated by many genes such as BRCA1/2, PTEN, TP53, mTOR, TERT, AKT, PI3K and others genes. Treatment options for BC remain a hurdle, which warrants a comprehensive understanding that establishes an interlinking connection between these genes in BC tumorigenesis. Consequently, there is an increasing demand for alternative treatment approaches and the design of more effective treatments. In this regard, it is crucial to build the corresponding transcriptional regulatory networks governing BC by using advanced genetic tools and techniques. In the past, several molecular editing technologies have been used to edit genes with several limitations. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (CRISPR/Cas9) recently received wise attention due to its potential in biomedical and therapeutic applications. Here, we review the role of various molecular signalling pathways dysregulated in BC development such as PTEN/PI3K/AKT/mTOR as well as BRCA1/BRCA2/TP53/TERT and their interplay between the related gene networks in BC initiation, progression and development of resistance against available targeted therapeutic agents. Use of CRISPR/Cas9 gene-editing technology to generate BC gene-specific transgenic cell lines and animal models to decipher their role and interactions with other gene products has been employed successfully. Moreover, the significance of using CRISPR/Cas9 technology to develop early BC diagnostic tools and treatments is discussed here.

Breast cancer, menopause, and long-term survivorship: critical issues for the 21st century

Genome editing technology and applications with the type I CRISPR system

CRISPR-GE: A Convenient Software Toolkit for CRISPR-Based Genome Editing

SummaryThe clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system is an effective genome editing tool for plant and animal genomes. However, there are still few reports on the successful application of CRISPR–Cas9 to horticultural plants, especially with regard to germ‐line transmission of targeted mutations. Here, we report high‐efficiency genome editing in the wild strawberry Fragaria vesca and its successful application to mutate the auxin biosynthesis gene TAA1 and auxin response factor 8 (ARF8). In our CRISPR system, the Arabidopsis U6 promoter AtU6‐26 and the wild strawberry U6 promoter FveU6‐2 were each used to drive the expression of sgRNA, and both promoters were shown to lead to high‐efficiency genome editing in strawberry. To test germ‐line transmission of the edited mutations and new mutations induced in the next generation, the progeny of the primary (T0) transgenic plants carrying the CRISPR construct was analysed. New mutations were detected in the progeny plants at a high efficiency, including large deletions between the two PAM sites. Further, T1 plants harbouring arf8 homozygous knockout mutations grew considerably faster than wild‐type plants. The results indicate that our CRISPR vectors can be used to edit the wild strawberry genome at a high efficiency and that both sgRNA design and appropriate U6 promoters contribute to the success of genomic editing. Our results open up exciting opportunities for engineering strawberry and related horticultural crops to improve traits of economic importance.

Context: The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system is a groundbreaking gene-editing tool that shows great promise for modifying genomes. Derived from prokaryotic adaptive immune defense mechanisms, this technique has been used in research on human diseases, demonstrating remarkable therapeutic potential. Through CRISPR, specific genetic mutations in patients can be corrected during gene therapy, offering a solution for treating diseases that were previously untreatable using conventional methods. This review explores the recent progress and future prospects of the CRISPR system, focusing on its applications in medicine and stem cell engineering. Special emphasis is placed on medical applications, the latest target design or analysis tools for genome editing, advancements in stem cell engineering, and associated innovations and challenges. Evidence Acquisition: This study reviewed articles indexed in ISI, SID, PubMed, and PubMed Central from 2007 to 2024. Results: Cas9, a key protein in CRISPR gene editing, is an endonuclease capable of targeting and cutting specific DNA sequences, guided by short RNA sequences. The gene editing process involves homology-directed repair (HDR), non-homologous end joining (NHEJ), and base editing pathways. Base editing, which modifies the epigenome without inducing DNA breaks, is gaining increasing attention. However, CRISPR still faces technical challenges, and the development of more efficient "super" CRISPR technology will likely require time. This article reviews the effectiveness, limitations, and applications of the CRISPR system. Conclusions: CRISPR/Cas9 tools allow for the creation of precise models, leading to more effective treatment options for patients.

Simple SummaryThe technique of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has revolutionised cancer research, including breast cancer and triple negative breast cancer (TNBC). By employing this technique, scientists can now better model these diseases, discover unknown genes that play a role in cancer progression, facilitate a more sensitive and earlier diagnosis of breast cancer and triple negative breast cancer (TNBC), and even determine if there is the possibility of providing more selective and efficient treatments. To do so, scientists are trying to optimise the distribution of the CRISPR components in the tumour by using several methods that we have listed. In this work, we have also highlighted the weak points and the future perspectives that CRISPR possesses. Undoubtedly, CRISPR technology can improve many aspects of breast cancer/TNBC research.Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has transformed oncology research in many ways. Breast cancer is the most prevalent malignancy globally and triple negative breast cancer (TNBC) is one of the most aggressive subtypes with numerous challenges still to be faced. In this work, we have explained what CRISPR consists of and listed its applications in breast cancer while focusing on TNBC research. These are disease modelling, the search for novel genes involved in tumour progression, sensitivity to drugs and immunotherapy response, tumour fitness, diagnosis, and treatment. Additionally, we have listed the current delivery methods employed for the delivery of CRISPR systems in vivo. Lastly, we have highlighted the limitations that CRISPR technology is subject to and the future directions that we envisage. Overall, we have provided a round summary of the aspects concerning CRISPR in breast cancer/TNBC research.

Does a young woman living with a smoker or taking a job working in a smoky bar have a greater chance of developing breast cancer? Some scientists believe that such situations can indeed raise a woman’s risk of developing breast cancer before the age of 50. Because epidemiological and toxicological studies show that women’s breast tissue may be especially sensitive to exposure to carcinogens prior to first pregnancy, these researchers contend that public education should be directed at alerting adolescents and young women to the potential risk. However, not everyone in the international public health community agrees that the evidence to date supports a link between passive smoking and breast cancer, and some say that women are being alarmed unnecessarily. This disagreement has sparked debate that is sometimes heated. The stakes are high because breast cancer is the most common cancer in women in industrialized countries, according to the WHO. It is the leading cancer killer of nonsmoking women, and second only to lung cancer deaths among women who smoke. Among the researchers interviewed for this article who disagree that there is enough evidence to link secondhand smoke (SHS) with breast cancer, the majority call the evidence to date “suggestive but not sufficient,” as the Surgeon General’s 2006 report, The Health Consequences of Involuntary Exposure to Tobacco Smoke, put it. That characterization is based largely on the fact that the research considered when the Surgeon General’s report was being amassed did not clearly link even active smoking to breast cancer. Researchers in this camp do, however, stress that ongoing campaigns to prohibit smoking in public will protect the whole of society against the wide variety of ills proven to be caused by SHS. These include lung cancer, cardiovascular disease, and sudden infant death syndrome, among others. A smaller group contends that the question of whether or not SHS causes breast cancer is a political issue with the potential to compromise the scientific process. “A premature decision about causality could jeopardize the credibility of the entire review process and all of the other, established effects of secondhand smoke,” says Michael Thun, national vice president of epidemiology and surveillance research for the American Cancer Society. Adds Valerie Beral, director of the University of Oxford Cancer Research UK Epidemiology Unit, “To prematurely come to conclusions about the causation when there is a big division in the scientific community . . . is bad science.” Thun debated the subject in a series of public forums held in conjunction with scientific meetings. Taking the opposing view was Kenneth C. Johnson, a research scientist with the Public Health Agency of Canada, who was one of the first scientists to discern a potential link. During the debates, Johnson pointed out there are about the same number of studies linking breast cancer to passive smoking as there were linking lung cancer to SHS in 1986, when the Surgeon General concluded that passive smoking caused lung cancer. Johnson also says that more of the breast cancer studies are statistically significant, and that the estimated risk for breast cancer is higher.

Genome editing technology has emerged as a potential therapeutic tool for treating incurable diseases. In particular, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems and the design of single-guide RNAs (sgRNAs) have revolutionized genome editing applications. Unfortunately, compared with the rapid development of gene-editing tools, the progress in the development of delivery technologies is lagging behind and thus limiting the clinical application of genome editing. To overcome these limitations, researchers have investigated various delivery systems, including viral and non-viral vectors for delivering CRISPR/Cas and sgRNA complexes. As natural endogenous nanocarriers, extracellular vesicles (EVs) present advantages of biocompatibility, low immunogenicity, stability, and high permeability, making them one of the most promising drug delivery vehicles. This review provides an overview of the fundamental mechanisms of EVs from the aspects of biogenesis, trafficking, cargo delivery, and function as nanotherapeutic agents. We also summarize the latest trends in EV-based CRISPR/Cas delivery systems and discuss the prospects for future development. In particular, we put our emphasis on the state-of-the-art engineering strategies to realize efficient cargo packaging and loading. Altogether, EVs hold promise in bridging genome editing in the laboratory and clinical applications of gene therapies by providing a safe, effective, and targeted delivery vehicle.

CRISPR-based adaptive and heritable immunity in prokaryotes

In It Together