CRISPR-Cas9 Gene Editing Research Articles

Strategies of Bladder Reconstruction after Partial or Radical Cystectomy for Bladder Cancer.

The standard strategy is to reconstruct bladder by use of bowel segments as material in bladder cancer with radical cystectomy clinically. Both natural derived and non natural derived materials are investigated in bladder reconstruction. Studies on mechanical bladder, bladder transplantation and bladder xenotransplantation are currently limited although heart and kidney transplantation or xenotransplantation are successful to a certain extent, and bone prostheses are applied in clinical contexts. Earlierlimited number of studies associated with bladder xenograft from animals to humans were notparticular promisingin results. Although there have beeninvestigations on pig to human cardiac xenotransplantation with CRISPR Cas9 gene editing, theCRISPR Cas techniqueisnot yet widelyresearchedin porcinebladder related gene editingfor the potential of human bladder replacementfor bladder cancer. The advancement of technologies such as gene editing, bioprinting and induced pluripotent stem cells allow further research into partial or wholebladder replacement strategies. Porcine bladder is suggested as a potential source material for bladder reconstruction due to its alikeness to human bladder. Challenges thatexist with all these approaches need to be overcome. This paper aims to review gene editing technology such as the CRISPR Cas systems as tools in bladder reconstruction, bladder xenotransplantation and hybrid bladder with technologies of induced pluripotent stem cells and genome editing, bioprinting for bladder replacement for bladder reconstruction and to restore normal bladder control function after cystectomy for bladder cancer.

Frameworks for transformational breakthroughs in RNA-based medicines.

RNA has sparked a revolution in modern medicine, with the potential to transform the way we treat diseases. Recent regulatory approvals, hundreds of new clinical trials, the emergence of CRISPR gene editing, and the effectiveness of mRNA vaccines in dramatic response to the COVID-19 pandemic have converged to create tremendous momentum and expectation. However, challenges with this relatively new class of drugs persist and require specialized knowledge and expertise to overcome. This Review explores shared strategies for developing RNA drug platforms, including layering technologies, addressing common biases and identifying gaps in understanding. It discusses the potential of RNA-based therapeutics to transform medicine, as well as the challenges associated with improving applicability, efficacy and safety profiles. Insights gained from RNA modalities such as antisense oligonucleotides (ASOs) and small interfering RNAs are used to identify important next steps for mRNA and gene editing technologies.

Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation

Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

Gene Editing for CEP290-Associated Retinal Degeneration

BackgroundCEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant).MethodsWe performed a phase 1–2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora–Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire–25 (in adults) or the Children’s Visual Function Questionnaire (in children).ResultsEDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log10 of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score.ConclusionsThe safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1–2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes. (Funded by Editas Medicine and others; BRILLIANCE ClinicalTrials.gov number, NCT03872479.)

Tumor Microenvironment-Responsive Nanocapsule Delivery CRISPR/Cas9 to Reprogram the Immunosuppressive Microenvironment in Hepatoma Carcinoma.

Cancer immunotherapy has demonstrated significant efficacy in various tumors, but its effectiveness in treating Hepatocellular Carcinoma (HCC) remains limited. Therefore, there is an urgent need to identify a new immunotherapy target and develop corresponding intervention strategies. Bioinformatics analysis has revealed that growth differentiation factor 15 (GDF15) is highly expressed in HCC and is closely related to poor prognosis of HCC patients. The previous study revealed that GDF15 can promote immunosuppression in the tumor microenvironment. Therefore, knocking out GDF15 through gene editing could potentially reverse the suppressive tumor immune microenvironment permanently. To deliver the CRISPR/Cas9 system specifically to HCC, nanocapsules (SNC) coated with HCC targeting peptides (SP94) on their surface is utilized. These nanocapsules incorporate disulfide bonds (SNCSS) that release their contents in the tumor microenvironment characterized by high levels of glutathione (GSH). In vivo, the SNCSS target HCC cells, exert a marked inhibitory effect on HCC progression, and promote HCC immunotherapy. Mechanistically, CyTOF analysis showed favorable changes in the immune microenvironment of HCC, immunocytes with killer function increased and immunocytes with inhibitive function decreased. These findings highlight the potential of the CRISPR-Cas9 gene editing system in modulating the immune microenvironment and improving the effectiveness of existing immunotherapy approaches for HCC.

Viral Delivery of Compact CRISPR-Cas12f for Gene Editing Applications.

Treating human genetic conditions invivo requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with ex vivo therapies and with invivo delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body. However, the large size of most CRISPR-Cas systems limits packaging into the viral genome and reduces drug development flexibility and manufacturing efficiency. Here, we demonstrate efficient CRISPR gene editing using a miniature CRISPR-Cas12f system with expanded genome targeting packaged into AAV particles. We identified efficient guides for four therapeutic gene targets and encoded the guides and the Cas12f nuclease into a single AAV. We then demonstrate editing in multiple cell lines, patient fibroblasts, and primary hepatocytes. We then screened the cells for off-target editing, demonstrating the safety of the therapeutics. These results represent an important step in applying CRISPR editing to diverse genetic sequences and organs in the body.

Generating CRISPR Alleles in Drosophila.

CRISPR gene editing is a versatile and efficient approach for generating a wide variety of genetic reagents in flies. This unparalleled ability to manipulate the genome has revolutionized neuroscience, allowing Drosophila neurobiologists to readily generate new alleles to probe gene function, investigate the functional consequences of disease-associated variants, tag endogenous proteins to follow their dynamic localization in neurons and glia, and much more. Here, we provide a comprehensive protocol for generating heritable mutations in Drosophila We particularly focus on design considerations and tips for avoiding common errors to maximize the likelihood of successful gene editing.

Introduction to CRISPR and Its Use in Drosophila.

The preeminence of Drosophila genetics has led to key discoveries in biology across a variety of fields and disciplines. The advent of CRISPR gene editing has expanded the toolkit of genetic reagents that can be applied to manipulate and observe genes, RNAs, and proteins in an in vivo context. This review describes CRISPR and its use as a transformative gene editing tool in Drosophila We focus on the canonical pathway in which the Cas9 nuclease is directed to specific sequences by guide RNA (gRNA), where cleavage leads to DNA repair by one of two main cellular pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The error-prone NHEJ pathway can be appropriated to disrupt targeted sequences, enabling a variety of loss-of-function studies. Induction of the HDR pathway allows precise editing, including defined deletions, the introduction of specific sequence changes, and the incorporation of fluorescent and epitope tags. These approaches have increased the power of Drosophila genetics and been successfully used to conduct in vivo structure-function studies, study disease-associated variants, and follow protein dynamics.

Abstract 2044: Disruption Of DENND5B Attenuates Triglyceride-rich Lipoprotein Secretion From Human Enterocytes And Hepatocytes

Background and Hypothesis: Elevated plasma triacylglycerides (TAGs) are an established risk factor for atherosclerotic cardiovascular disease, and are regulated by dietary absorption via the small intestine and endogenous production by the liver. Our laboratory formerly reported that Dennd5b -deficient mice are protected from diet-induced hepatic steatosis, hyperlipidemia, and atherosclerosis. Electron microscopy imaging of the intestinal epithelium of Dennd5b -/- mice reveals significant lipid accumulation in intracellular chylomicron secretory vesicles, suggesting a post-Golgi chylomicron secretion defect. Given that Dennd5b is expressed by the two major triglyceride-rich lipoprotein (TRL)-secreting organs (liver and small intestine), we hypothesized that disruption of DENND5B attenuates TRL secretion in both of these cell types in humans. Methods and Results: To determine if DENND5B disruption impairs TRL secretion in human cells, we generated DENND5B -/- intestinal epithelial (Caco-2) and hepatocyte (HepG2) cell lines using CRISPR gene editing. Western blots verified DENND5B protein reduction in DENND5B -/- cells. The impact of DENND5B deficiency on TRL secretion was quantified by measuring the cellular and secreted TAG content of DENND5B +/+ and DENND5B -/- cells under standard growth conditions and after oleic acid loading. Caco-2 cells were differentiated on membrane supports for 21 days prior to experiments. DENND5B -/- Caco-2 cells secreted significantly less TAGs compared to wild-type Caco-2 cells (-67.8%, p<0.0001). DENND5B -/- HepG2 cells also had significantly reduced TAG secretion (-91.0%, p<0.0001). Disruption of DENND5B in both cell lines reduced TAG secretion without affecting cellular TAG content. Immunofluorescence microscopy showed intracellular DENND5B protein localization consistent with secretory pathway involvement with detection on a perinuclear structure (likely Golgi), cytosolic vesicles, and plasma membrane. Conclusions: These studies suggest that DENND5B plays a role in TRL secretion from human enterocytes and hepatocytes and provide new model systems for investigating the molecular function of DENND5B in TRL secretion from human cells.

Pituitary Neuroendocrine Tumor Organoids Generated from Induced Pluripotent Stem Cells Reveal the Dysregulation of Cell Lineage Differentiation in Cushing’s Disease

Background: Cushing’s Disease (CD) is an endocrine disease typically caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET). Corticotroph subtype PitNETs subsequently stimulate the adrenal glands to overproduce causing a wide range of detrimental effects on health, including increased stroke rates, diabetes, obesity, depression, anxiety, and a threefold increase in the risk of death from cardiovascular disease. In some instances, CD is a manifestation of genetic mutation syndromes that include MEN1, FIPA, CDH23 and Carney complex. In other cases, somatic mutations underlie the development of CD including mutations in the deubiquitinase gene USP8, identified in 35-62 % of sporadic corticotroph PitNETs and USP48 as a driver mutation in USP8 wild-type tumors. There are currently no in vitro models to study the impact of genetic mutations on the pathways leading to PitNET development in humans. Objective: To define the cellular complexity of corticotroph subtype PitNETs at a spatial single-cell resolution. Methodology: Organoids were generated from a CD patient expressing a CDH23 mutation (hPitNETO iPSCDH23) as reported by our group (Mallick et al., 2023, Translational Research). CRISPR-Cas9 gene editing of iPSCs was also used to model the development of corticotroph PitNETs expressing USP48 and USP8 mutations. Human PitNET organoids (PitNETOs) were generated from tumor tissue harvested during transsphenoidal surgery. Cell lineage trajectory analysis was performed on scRNA-seq data collected during the iPSC differentiation program. ScRNA-seq was used to compare the transcriptomic profile and cell composition of CD patient PitNETOs to the patient’s own tissue using the NanoString CosMxTM spatial transcriptomic analysis. Results: Organoids generated from a CD patient expressing a CDH23 mutation (hPitNETO iPSCDH23) exhibited tumor behavior consistent with the expected pathology and function of sparsely granulated corticotroph PitNETs. Cell lineage trajectories of hPitNETO iPCCtrl control cultures revealed multiple branching paths from the stem-like (SOX2/PAX7+) base cluster leading to clusters enriched for markers of pituitary cell lineages (lactotroph, somatotroph, thyrotroph, gonadotroph and corticotroph). Trajectory analysis for hPitNETO iPSCDH23 showed dysregulation in the normal pituitary cell lineage differentiation leading to the development of poorly differentiated corticotrophs (PAX7+/TPit+) and PAX7+/stem cells. CosMxTM spatial gene mapping revealed a cell population co-expressing PAX7 and stem cell markers SOX2 and CD44 that was abundant in Crooke cell and sparsely granulated TPit+ PitNET subtypes. An increase in the PAX7/SOX2/CD44 PitNET cell population correlated with a higher Knosp grade, tumor size and invasiveness, when compared to densely granulated PitNETs. Conclusions: Defining the complexity of the PitNET microenvironment will provide insights into the pathogenesis of CD and potentially lead to the stratification of high-risk patients for disease recurrence. 1R01DK133325. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Knockout of DEPDC5, the gene associated with drug-resistant focal temporal epilepsy, causes abnormal Ca2+-waves in human iPS cell-induced neurons

Genetic variants in the DEP domain containing 5 (DEPDC5) are one of the most common causes of inheritable drug-resistant temporal focal epilepsy. Epilepsies caused by DEPDC5 variants are often associated with brain malformations and sudden unexplained death in epilepsy (SUDEP). It has been reported that the DEPDC5 genes regulates cell growth via rapamycin complex 1 (mTORC1) pathways. Though several animal models showed knockout of the DEPDC5 causes epilepsy, the underlying mechanism(s) by which DEPDC5 variants cause neuronal excitations remains unelucidated in human cells. To examine whether knockout of DEPDC5 causes abnormal excitations in human iPS cell (iPSC)-induced neurons, DEPDC5 was knocked out using CRISPR-Cas9 gene editing methods. Expression of DEPDC5 peptides was confirmed by a mass spectroscopy. Then, the cells were differentiated into neurons by Dual Smad Inhibition. Spontaneous cellular excitation was evaluated by Ca2+-imaging (AquaCosmos/Ratio imaging system, Hamamatsu Photonics). DNA sequencing of the DEPDC5-knockout cells showed a truncated change, c.251_258delCTTTGGGG:(p.Gly15Glyfs*5), in one allele. The DEPDC5 peptides encoded by exon 7 were decreased to less than half in the DEPDC5-knockout cells compared to the wild type (WT) cells. Ca2+-imaging showed abnormal Ca2+ bursts in neurons derived from the DEPDC5-knockout cells, while the WT cells did not show any abnormal Ca2+ waves. These results indicate that haploinsuffciency of DEPDC5 can cause abnormal excitation in human iPSC-induced neurons. Currently, we are planning to further investigate cellular excitations in iPSC-induced cardiomyocytes to dissect underlying mechanisms of SUDEP. This work was supported in part by a Grant-in-Aid for Scientific Research (C) [grant number 18K08900 and 21K08135 (to T.A.)] and a Grant-in-Aid for Young Scientists [grant number 22K15694 (to Y.H.)] from the Japan Society for the Promotion of Science. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.

We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

Ablation of specific insulin-like growth factor I forms reveals the importance of cleavage for regenerative capacity and glycosylation for skeletal muscle storage.

Insulin-like growth factor-I (IGF-I) facilitates mitotic and anabolic actions in all tissues. In skeletal muscle, IGF-I can promote growth and resolution of damage by promoting satellite cell proliferation and differentiation, suppressing inflammation, and enhancing fiber formation. While the most well-characterized form of IGF-I is the mature protein, alternative splicing and post-translational modification complexity lead to several additional forms of IGF-I. Previous studies showed muscle efficiently stores glycosylated pro-IGF-I. However, non-glycosylated forms display more efficient IGF-I receptor activation invitro, suggesting that the removal of the glycosylated C terminus is a necessary step to enable increased activity. We employed CRISPR-Cas9 gene editing to ablate IGF-I glycosylation sites (2ND) or its cleavage site (3RA) in mice to determine the necessity of glycosylation or cleavage for IGF-I function in postnatal growth and during muscle regeneration. 3RA mice had the highest circulating and muscle IGF-I content, whereas 2ND mice had the lowest levels compared to wild-type mice. After weaning, 4-week-old 2ND mice exhibited higher body and skeletal muscle mass than other strains. However, by 16 weeks of age, muscle and body size differences disappeared. Even though 3RA mice had more IGF-I stored in muscle in homeostatic conditions, regeneration was delayed after cardiotoxin-induced injury, with prolonged necrosis most evident at 5 days post injury (dpi). In contrast, 2ND displayed improved regeneration with reduced necrosis, and greater fiber size and muscle mass at 11 and 21 dpi. Overall, these results demonstrate that while IGF-I glycosylation may be important for storage, cleavage is needed to enable IGF-I to be used for efficient activity in postnatal growth and following acute injury.

Generation of a homozygous DNAJC19 knockout human embryonic stem cell line by CRISPR/Cas9 system

The DNAJC19 gene, a member of DNAJ heat shock protein (Hsp40) family, is localized within the inner mitochondrial membrane (IMM) and plays a crucial role in regulating the function and localization of mitochondrial Hsp70 (MtHsp70). Mutations in the DNAJC19 gene cause Dilated Cardiomyopathy with Ataxia Syndrome (DCMA). The precise mechanisms underlying the DCMA phenotype caused by DNAJC19 mutations remain poorly understood, and effective treatment modalities were lacking unitl recently. By using CRISPR-Cas9 gene editing technology, this study generated a DNAJC19-knockout (DNAJC19-KO) human embryonic stem cell line (hESC), which will be a useful tool in studying the pathogenesis of DCMA.

Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

The Application of CRISPR Gene Editing Technology in PET Biodegradation

Plastics used in people's daily life bring convenience on the one hand, and on the other hand, a large amount of plastic waste also brings great pollution to the environment. Polyethylene terephthalate (PET) is a polymer polymerization material, which has become one of the most widely used plastics in the world because of its durability and easy processing. The stability of the ecological environment around it is seriously threatened by the large amount of PET used and its difficult degradation.PET even endangered human health. The traditional methods of PET treatment are physical and chemical methods such as incineration, landfill, and heat cracking, etc. Comoared with the first two generations of gene-editing system, CRISPR/Cas9 system has the advantage of easy design, low cost and high efficiency. Currently, the most favorable method of treating PET to protect the environment is biodegradation. This paper introduces the nature of PET and the research progress of PET degradation enzymes, seeking to improve the activity and thermal stability of degradation enzymes by CRISPR/Cas9 gene editing technology, this paper explores the construction of mutants with high thermal stability and high degradation activity of the fusion dual enzyme of PETase and MHETase by CRISPR/Cas9 gene editing technology.

CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within noncoding regions. We have adapted the CRISPR-Cas9 gene editing tool as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks. Whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002) showed an average of 417 somatic PAMs per tumor produced from single base substitutions. Further analyses of 591 paired T-N samples from The International Cancer Genome Consortium found medians of ∼455 somatic PAMs per tumor in pancreatic, ∼2800 in lung, and ∼3200 in esophageal cancer cohorts. Finally, we demonstrated 69-99% selective cell death of three targeted pancreatic cancer cell lines using 4-9 sgRNAs designed using the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs in either the patient's normal cells or an irrelevant cancer using WGS. This study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy, and supports the genetic targeting of adult cancers.

Abstract LE21: Mechanisms of gene regulation by chromatin adaptors in development and disease

Abstract Complex biological processes, such as organismal development, are directly influenced by the interplay between genes and the cellular structures and environmental factors that regulate them. Key players in this regulation are chromatin adaptor proteins, which can impact the genetic and epigenetic programs of cells. However, our understanding of how these proteins interact with chromatin - the physiological form of our genomes - and regulate gene activity is limited, especially in the context of diseases like cancer. To test our central hypothesis and to understand how chromatin adaptors contribute to cell function, we are developing innovative high-throughput and biochemical approaches. These will help us systematically identify specific histone modifications that affect the binding of Menin and other adaptors to chromatin. Our experimental approach includes profiling, validation, and functional characterization using advanced genomic and epigenomic technologies, transcriptional profiling, and CRISPR gene editing. The application of these techniques will reveal, for the first time, the molecular mechanisms by which these proteins bind to specific parts of the genome and regulate gene activity. Our findings will enhance our understanding of how chromatin adaptors, like Menin, interact with chromatin and regulate gene activity in different tissues, and how these functions are disrupted by cancer-associated mutations. This will also contribute to the development of potential new therapies that target these altered mechanisms. Furthermore, our findings can be extended to other proteins involved in chromatin and epigenetic regulation, enabling a broader application in understanding and treating cancer and other human diseases. Citation Format: Yadira Marie Soto-Feliciano. Mechanisms of gene regulation by chromatin adaptors in development and disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LE21.

Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness

BackgroundTo successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4.MethodsWe characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4.ResultsTrx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice.ConclusionsTrx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.Graphical

(275) - An In Vitro Study of Virus-Like Particle Mediated CRISPR Gene Editing to Support Gene Modulation in the Human Ex Vivo Lung

(275) - An In Vitro Study of Virus-Like Particle Mediated CRISPR Gene Editing to Support Gene Modulation in the Human Ex Vivo Lung