High-throughput Genetic Screens Research Articles

Mitofusin-mediated contacts between mitochondria and peroxisomes regulate mitochondrial fusion.

Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.

Abstract 6988: TP53RK regulates DNA replication by CDC7-mediated MCM helicase phosphorylation in colorectal cancer

Abstract Background: We identify TP53 regulating kinase (TP53RK), which showed the highest cell growth inhibition efficiency in six colon cancer cell lines. TP53RK is overexpressed in various cancer types, including multiple myeloma and skin cancer, but the mechanism for its tumorigenesis in colon cancer is unknown. Therefore, this study aims to reveal the tumorigenesis mechanism of TP53RK, which is overexpressed in Colorectal Cancer (CRC), through global and phospho-proteomics. Method: A total 5 CRC cell lines (HT29, SW480, HCT116, H508 and CaCO2), colon normal fibroblast CRL1459, HCT116 p53 null cell and patient-derived normal organoid were used. Immunoblotting, MTT assay, Colony formation assay, Annexin-V assay and cell cycle analysis were performed for evaluation of TP53RK loss-of-function. With kinase-substrate enrichment analysis with phospho-proteome analysis, MCM2 was found to be a substrate for the kinase regulated by TP53RK. Results: High-throughput genetic screening approach have been widely applied to the study the gene function and molecular mechanism associated with tumorigenesis. By using genome-wide CRISPR/Cas9 library screening, we identify TP53RK, the first committed negative-selected gene, as a tyrosine kinase in the colorectal cancer(CRC) specific biomarker. Through proteome analysis, we found that depletion of TP53RK can hypo-phosphorylate the DNA helicase complex, MCM2 (minichromosome maintenance protein2). According to previous studies, the phosphorylation of MCM2 is regulated by CDC7 (cell division cycle7), and the abnormal states of CDC7 and MCM2 cause DNA replication disorder. Our results show that TP53RK expression is upregulated in CRC and its depletion results in decreased CDC7 expression and hypo-phosphorylation of the MCM complex. Decreased CDC7 expression due to its instability after TP53RK depletion can lead to DNA replication errors. DNA replication errors can lead cancer cells to apoptosis, suggesting that they may be a strategy for cancer treatment. In summary, we provide TP53RK as a novel prognostic and therapeutic target in CRC. Conclusion: To recapitulate briefly, our findings suggest that the interaction of TP53RK with CDC7 can regulate CDC7 protein stability and stabilized CDC7 can phosphorylate MCM2 to initiate DNA replication. In other words, because overexpression of TP53RK acts as an oncogene that promotes tumor progression in CRC, depletion of TP53RK can degrade CDC7 and induce colon cancer cells to death or regression. Citation Format: Younghee Choi, Ji-won Park, Jun-kyu Kang, Eun-ju Kim, Sang-Hyun Song, Eugene C. Yi, Tae-You Kim. TP53RK regulates DNA replication by CDC7-mediated MCM helicase phosphorylation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6988.

Expanding the flexibility of base editing for high-throughput genetic screens in bacteria.

Genome-wide screens have become powerful tools for elucidating genotype-to-phenotype relationships in bacteria. Of the varying techniques to achieve knockout and knockdown, CRISPR base editors are emerging as promising options. However, the limited number of available, efficient target sites hampers their use for high-throughput screening. Here, we make multiple advances to enable flexible base editing as part of high-throughput genetic screening in bacteria. We first co-opt the Streptococcus canis Cas9 that exhibits more flexible protospacer-adjacent motif recognition than the traditional Streptococcus pyogenes Cas9. We then expand beyond introducing premature stop codons by mutating start codons. Next, we derive guide design rules by applying machine learning to an essentiality screen conducted in Escherichia coli. Finally, we rescue poorly edited sites by combining base editing with Cas9-induced cleavage of unedited cells, thereby enriching for intended edits. The efficiency of this dual system was validated through a conditional essentiality screen based on growth in minimal media. Overall, expanding the scope of genome-wide knockout screens with base editors could further facilitate the investigation of new gene functions and interactions in bacteria.

Abstract A102: ROR2/Wnt5a signaling regulates directional cell migration and early tumor cell invasion in ovarian cancer

Abstract Adhesion to and clearance of the mesothelial monolayer are key events in the early metastatic seeding of ovarian cancer. To identify mediators of ovarian cancer cell adhesion and invasion, we developed and conducted a cellular, high-throughput genetic screen using a human kinome siRNA library. Through this screen, we identified ROR2 as a driver of ovarian cancer adhesion and invasion. ROR2 is a receptor tyrosine kinase that interacts with Wnt5a ligand to activate non-canonical Wnt signaling and has been previously shown to be upregulated in ovarian cancer tissue. However, no prior study has evaluated the mechanistic role of ROR2 in ovarian cancer. Using in vitro adhesion and mesothelial clearance assays, we show that ROR2 promotes ovarian cancer cell adhesion and clearance of the mesothelial monolayer, key early steps in ovarian cancer metastasis. Further, we show that depletion of ROR2 reduces metastatic tumor burden in a syngeneic mouse model of ovarian cancer. Additionally, using a microfluidic device capable of producing stable fluid gradients, we demonstrate that ROR2 expression in ovarian cancer cells serves to drive directed cell migration of tumor cells, preferentially to areas of high Wnt5a ligand. As Wnt5a has been previously shown to be produced and upregulated in the mesothelial cells of the ovarian cancer microenvironment, these findings represent a novel role for ROR2 to promote the homing of ovarian tumor cells to the mesothelial lined metastatic niche by using Wnt5a as a chemotactic cue. These findings support the role of ROR2 as a critical factor contributing to the steps of ovarian cancer metastasis. Therapeutic targeting of the ROR2/Wnt5a signaling axis could provide a means of improving treatment for patients with advanced ovarian cancer. Citation Format: Whitney R. Grither, Breanna Baker, Vasilios A. Morikis, Katherine C. Fuh, Gregoery D Longmore. ROR2/Wnt5a signaling regulates directional cell migration and early tumor cell invasion in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A102.

A novel de novo truncating variant in a Hungarian patient with CTNNB1 neurodevelopmental disorder

PurposeWe aimed to elucidate the underlying disease in a Hungarian family, with only one affected family member, a 16-year-old male Hungarian patient, who developed global developmental delay, cognitive impairment, behavioral problems, short stature, intermittent headaches, recurrent dizziness, strabismus, hypermetropia, complex movement disorder and partial pituitary dysfunction. After years of detailed clinical investigations and careful pediatric care, the exact diagnosis of the patient and the cause of the disease was still unknown.MethodsWe aimed to perform whole exome sequencing (WES) in order to investigate whether the affected patient is suffering from a rare monogenic disease.ResultsUsing WES, we identified a novel, de novo frameshift variant (c.1902dupG, p.Ala636SerfsTer12) of the catenin beta-1 (CTNNB1) gene. Assessment of the novel CTNNB1 variant suggested that it is a likely pathogenic one and raised the diagnosis of CTNNB1 neurodevelopmental disorder (OMIM 615,075).ConclusionsOur manuscript may contribute to the better understanding of the genetic background of the recently discovered CTNNB1 neurodevelopmental disorder and raise awareness among clinicians and geneticists. The affected Hungarian family demonstrates that based on the results of the clinical workup is difficult to establish the diagnosis and high-throughput genetic screening may help to solve these complex cases.

Fully genotyping and screening of clinically important blood-group antigens by MALDI TOF mass spectrometry.

Several molecular biology methods are available for high-throughput blood typing. In this study, we aimed to build a high-throughput blood-group genetic screening system for high-frequency blood-group antigen-negative rare-blood groups in donors and patients. The amplification primers for all blood-type gene fragments involving the selected alleles were designed for detection. Single-base extend primers were also designed based on specific loci. DNA fragments were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) for the last nucleotide identification of amplification products in the extend step. The accuracy was verified by known samples. Thirty-six random samples were detected by serological tests and sequencing to verify the system stability. After verification, according to the collected known rare-blood-type samples, all the alleles designed to be detected matched with the validated single-nucleotide polymorphisms. The verification tests showed that all genotyping results of the random samples were in accordance with the findings of serotyping and sequencing. Then, 1258 random donor samples were screened by the built typing system after the verification. Three Fy(a-) and four s- were screened out in 1258 random blood samples. The multiple polymerase chain reaction-based MS detection system can be used in rare-blood-type screening with good accuracy and stability.

The Current Situation and Development Prospect of Whole-Genome Screening.

High-throughput genetic screening is useful for discovering critical genes or gene sequences that trigger specific cell functions and/or phenotypes. Loss-of-function genetic screening is mainly achieved through RNA interference (RNAi), CRISPR knock-out (CRISPRko), and CRISPR interference (CRISPRi) technologies. Gain-of-function genetic screening mainly depends on the overexpression of a cDNA library and CRISPR activation (CRISPRa). Base editing can perform both gain- and loss-of-function genetic screening. This review discusses genetic screening techniques based on Cas9 nuclease, including Cas9-mediated genome knock-out and dCas9-based gene activation and interference. We compare these methods with previous genetic screening techniques based on RNAi and cDNA library overexpression and propose future prospects and applications for CRISPR screening.

Towards Understanding Neurodegenerative Diseases: Insights from Caenorhabditis elegans.

The elevated occurrence of debilitating neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD) and Machado-Joseph disease (MJD), demands urgent disease-modifying therapeutics. Owing to the evolutionarily conserved molecular signalling pathways with mammalian species and facile genetic manipulation, the nematode Caenorhabditis elegans (C. elegans) emerges as a powerful and manipulative model system for mechanistic insights into neurodegenerative diseases. Herein, we review several representative C. elegans models established for five common neurodegenerative diseases, which closely simulate disease phenotypes specifically in the gain-of-function aspect. We exemplify applications of high-throughput genetic and drug screenings to illustrate the potential of C. elegans to probe novel therapeutic targets. This review highlights the utility of C. elegans as a comprehensive and versatile platform for the dissection of neurodegenerative diseases at the molecular level.

Automated, high-throughput quantification of EGFP-expressing neutrophils in zebrafish by machine learning and a highly-parallelized microscope.

Normal development of the immune system is essential for overall health and disease resistance. Bony fish, such as the zebrafish (Danio rerio), possess all the major immune cell lineages as mammals and can be employed to model human host response to immune challenge. Zebrafish neutrophils, for example, are present in the transparent larvae as early as 48 hours post fertilization and have been examined in numerous infection and immunotoxicology reports. One significant advantage of the zebrafish model is the ability to affordably generate high numbers of individual larvae that can be arrayed in multi-well plates for high throughput genetic and chemical exposure screens. However, traditional workflows for imaging individual larvae have been limited to low-throughput studies using traditional microscopes and manual analyses. Using a newly developed, parallelized microscope, the Multi-Camera Array Microscope (MCAM™), we have optimized a rapid, high-resolution algorithmic method to count fluorescently labeled cells in zebrafish larvae in vivo. Using transgenic zebrafish larvae, in which neutrophils express EGFP, we captured 18 gigapixels of images across a full 96-well plate, in 75 seconds, and processed the resulting datastream, counting individual fluorescent neutrophils in all individual larvae in 5 minutes. This automation is facilitated by a machine learning segmentation algorithm that defines the most in-focus view of each larva in each well after which pixel intensity thresholding and blob detection are employed to locate and count fluorescent cells. We validated this method by comparing algorithmic neutrophil counts to manual counts in larvae subjected to changes in neutrophil numbers, demonstrating the utility of this approach for high-throughput genetic and chemical screens where a change in neutrophil number is an endpoint metric. Using the MCAM™ we have been able to, within minutes, acquire both enough data to create an automated algorithm and execute a biological experiment with statistical significance. Finally, we present this open-source software package which allows the user to train and evaluate a custom machine learning segmentation model and use it to localize zebrafish and analyze cell counts within the segmented region of interest. This software can be modified as needed for studies involving other zebrafish cell lineages using different transgenic reporter lines and can also be adapted for studies using other amenable model species.

Therapeutic Targeting of JAK/STAT Signaling and Histone Acetyltransferase Activity in Post-Myeloproliferative Neoplasm Secondary AML

Post-myeloproliferative neoplasm secondary Acute Myeloid Leukemia (Post-MPN sAML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms (MPN). Hyperactivation of JAK/STAT signaling is present in 50-90% of MPNs, underscoring its role in driving MPN pathogenesis and transformation to acute sAML. Ruxolitinib, a Type-I JAK2 inhibitor approved for the treatment of myelofibrosis, was tested in a Phase II clinical trial in post-MPN sAML. Despite enhancing patients' quality of life, ruxolitinib did not exert major improvements on disease outcome (Eghtedar et al, 2012). Thus, there is an urgent need to find synergistic drug combinations capable of complementing the therapeutic activity of JAK2 inhibitors in post-MPN sAML. Here, we demonstrate that combination treatment of ruxolitinib and CBP30, a bromodomain inhibitor of histone acetyltransferases (HAT) CREBBP and p300, enhances the therapeutic activity of ruxolitinib in post-MPN sAML using high throughput genetic screens, in vitro functional assays, transcriptomic and epigenetic profiling and mouse xenograft in vivo experiments. To identify druggable determinants of JAK2 inhibitor response in post-MPN sAML, we performed genome-wide loss-of-function CRISPR screens in sAML HEL cells treated with four different JAK2 inhibitors, including ruxolitinib. We discovered that depletion of the histone acetyl transferase CREBBP sensitizes HEL cells to JAK2 inhibition (Figure 1A). Pharmacological inhibition of JAK/STAT signaling and HAT activity using a combination of ruxolitinib and CREBBP inhibitors significantly reduced the proliferation of sAML cells HEL and SET2 (Figure 1B). Among the CREBBP inhibitors, CBP30 synergizes best with ruxolitinib having Chou-Talalay combination indices of 0.152 and 0.704 in HEL and SET2, respectively. Moreover, combination treatment of ruxolitinib and CBP30 markedly increased apoptosis and induced cell cycle arrest at G1 in HEL. To mechanistically characterize the synergism between ruxolitinib and CBP30, we performed ChIP-seq to profile STAT5 binding and H3K27ac levels in HEL cells treated with DMSO, ruxolitinib, CBP30 and their combination. STAT5 binding profiles did not reveal major differences in ruxolitinib versus ruxolitinib + CBP30 treated cells, suggesting that the drug combination does not significantly alter STAT5 binding. Conversely, we noticed striking global changes in H3K27ac levels in the combination compared with DMSO or single agents, indicating that the synergistic effect between ruxolitinib and CBP30 is mediated by substantial changes in transcriptional regulation. Notably, analysis of H3K27ac downregulated and upregulated sites identified enrichments in motifs recognized by FOS/SMAD4 and GATA transcription factors, respectively. These TFs are involved in the regulation of myeloid differentiation implicating a synergistic regulation of relevant myeloid differentiation pathways by the combination of JAK2 and CREBBP inhibitors in sAML. Furthermore, ruxolitinib + CBP30 treatment in HEL elicits a unique transcriptome profile with expression deregulation of genes associated with cell cycle, DNA repair, HAT activity and TGF-beta signaling as determined by DAVID functional annotation analysis. Lastly, to evaluate the synergistic effects of ruxolitinib and CBP30 in vivo, we treated luciferase-expressing HEL sAML mouse xenografts with vehicle, ruxolitinib (80 mg/kg), CBP30 (30 mg/kg) and ruxolitinib (80 mg/kg) + CBP30 (30 mg/kg). Combination of ruxolitinib and CBP30 displayed significant decrease in leukemia burden after 4 weeks of treatment as monitored by bioluminescence imaging and quantification. Consistently, mice treated with both drugs also showed remarkable decrease in their white blood cell count and spleen size compared with single-agent-treated and vehicle-treated mice. Collectively, our results substantiate the combinatorial therapeutic targeting of JAK/STAT signaling and HAT activity as a potential treatment approach for post-MPN sAML.

CRISPR-Cas System Is an Effective Tool for Identifying Drug Combinations That Provide Synergistic Therapeutic Potential in Cancers.

Despite numerous efforts, the therapeutic advancement for neuroblastoma and other cancer treatments is still ongoing due to multiple challenges, such as the increasing prevalence of cancers and therapy resistance development in tumors. To overcome such obstacles, drug combinations are one of the promising applications. However, identifying and implementing effective drug combinations are critical for achieving favorable treatment outcomes. Given the enormous possibilities of combinations, a rational approach is required to predict the impact of drug combinations. Thus, CRISPR-Cas-based and other approaches, such as high-throughput pharmacological and genetic screening approaches, have been used to identify possible drug combinations. In particular, the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful tool that enables us to efficiently identify possible drug combinations that can improve treatment outcomes by reducing the total search space. In this review, we discuss the rational approaches to identifying, examining, and predicting drug combinations and their impact.

Characteristics of hearing loss-associated gene mutations: A multi-center study of 119,606 neonates in Gannan.

HL is the second most common congenital disability in China, and its high incidence brings a serious burden of medical and educational sequelae. HL genetic screening enables the identification of individuals with inherited HL and carriers in a large scale. This study aimed to measure the detection rates of hearing loss (HL)-associated gene mutations in the Gannan population. The molecular etiology and risk factors of hereditary HL were also analyzed. In total, 119,606 newborns from 18 districts of Gannan were enrolled in this multi-center study conducted between April 2019 and April 2021. Otoacoustic Emission (OAE) was used for primary hearing screening 3 days after birth in quiet conditions, and OAE combined with automated auditory brainstem response (AABR) was applied 29-42 days after birth for those who failed or missed the initial screening. Meanwhile, high-throughput sequencing of hotspot HL-associated mutations in GJB2, GJB3, MTRNR1, and SLC26A4 were performed. Among the 119,606 newborns, 7796 (6.52%) failed the hearing screening. Genetic screening revealed that 5092 neonates (4.26%) carried HL-associated mutations. The detection rate of GJB2, SLC26A4, MTRNR1 and GJB3 mutations were 2.09%, 1.51%, 0.42% and 0.24%, respectively. The most prevalent variant was GJB2 c.235delC (1.74%). The second most prevalent variant was SLC26A4 c.919-2A>G (0.93%). The population who failed the hearing screening had a lower proportion (24.64%) of SLC26A4 gene variants compared to the population who passed (37.46%). Genetic screening identified 4612 (3.86%) carriers who were normal in hearing screenings. The concurrent hearing and genetic screening identified 480 (0.40%) neonates at high risk for hereditary HL. The results of this study suggest that the concurrent hearing screening and high-throughput genetic screening would greatly improve the effectiveness of newborn HL programs. This integration also facilitates the management of congenital HL, and aids in the prevention of aminoglycoside antibiotics-induced HL.

Automating high-throughput screening for anthracnose resistance in common bean using allele specific PCR

BackgroundCommon beans (Phaseolus vulgaris L.) provide important protein and calories globally. Anthracnose (Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, 1889) is a major disease in common bean and causes significant yield losses in bean production areas. Screening for markers linked to known disease resistance genes provides useful information for plant breeders to develop improved common bean varieties. The Kompetitive Allele Specific PCR (KASP) assay is an affordable genetic screening technique that can be used to accelerate breeding programs, but manual DNA extraction and KASP assay preparation are time-consuming. Several KASP markers have been developed for genes involved in resistance to bean anthracnose, which can reduce yield by up to 100%, but their usefulness is hindered by the labor required to screen a significant number of bean lines. Our research objective was to develop publicly available protocols for DNA extraction and KASP assaying using a liquid handling robot (LHR) which would facilitate high-throughput genetic screening with less active human time required. Anthracnose resistance markers were used to compare manual and automated results.ResultsThe 12 bean anthracnose differential cultivars were screened for four anthracnose KASP markers linked to the resistance genes Co-1, Co-3 and Co-42 both by hand and with the use of an LHR. A protocol was written for DNA extraction and KASP assay thermocycling to implement the LHR. The LHR protocol reduced the active human screening time of 24 samples from 3h44 to 1h23. KASP calls were consistent across replicates but not always accurate for their known linked resistance genes, suggesting more specific markers still need to be developed. Using an LHR, information from KASP assays can be accumulated with little active human time.ConclusionResults suggest that LHRs can be used to expedite time-consuming and tedious lab work such as DNA extraction or PCR plate filling. Notably, LHRs can be used to prepare KASP assays for large sample sizes, facilitating higher throughput use of genetic marker screening tools.

FLASH Genome Editing Pipeline: An Efficient and High-Throughput Method to Construct Arrayed CRISPR Library for Plant Functional Genomics.

CRISPR/Cas9 genome editing is a revolutionary technology for plant functional genomics and crop breeding. In this system, the Cas9 nuclease is directed by a guide RNA (gRNA) to cut the DNA target and introduce mutation through error-prone DNA break repair. Owing to its simplicity, CRISPR/Cas9-mediated targeted gene knockout is widely used for high-throughput genetic screening in animal cell cultures and bacteria. However, high-throughput genetic screening using CRISPR/Cas9 is still challenging in plants. We recently established a new approach, named the FLASH genome editing pipeline, to construct an arrayed CRISPR library in plants. In this pipeline, a set of 12 PCR fragments with different lengths (referred to as FLASH tags) are used to index the Cas9/gRNA vectors. Subsequently, a mixture of 12 Agrobacterium strains, in which each strain contained a FLASH-tag indexed vector, was transformed into rice plants. As a result, a unique link between the target gene/gRNA and FLASH tag is generated, which allows reading gRNA information in bacterial strains and gene-edited plants using regular PCR and gel electrophoresis. This protocol includes step-by-step instructions for gRNA design, high throughput assembly of FLASH-tag indexed Cas9/gRNA plasmids, Agrobacterium-mediated transformation of 12 indexed plasmids, and fast assignment of target gene information in primary transformants. The arrayed CRISPR library described here is suitable for small- to large-scale genetic screening and allows fast and comprehensive gene function discovery in plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assembly of FLASH-tag-indexed Cas9/gRNA plasmids Basic Protocol 2: Preparation of the Cas9/gRNA plasmid library Basic Protocol 3: Library preparation of Agrobacterium strains and mixing FLASH-tag indexed strains Basic Protocol 4: Grouped transformation and assignments of gRNA information of gene-edited plants.

GRNA-SeqRET: a universal tool for targeted and genome-scale gRNA design and sequence extraction for prokaryotes and eukaryotes.

High-throughput genetic screening is frequently employed to rapidly associate gene with phenotype and establish sequence-function relationships. With the advent of CRISPR technology, and the ability to functionally interrogate previously genetically recalcitrant organisms, non-model organisms can be investigated using pooled guide RNA (gRNA) libraries and sequencing-based assays to quantitatively assess fitness of every targeted locus in parallel. To aid the construction of pooled gRNA assemblies, we have developed an in silico design workflow for gRNA selection using the gRNA Sequence Region Extraction Tool (gRNA-SeqRET). Built upon the previously developed CCTop, gRNA-SeqRET enables automated, scalable design of gRNA libraries that target user-specified regions or whole genomes of any prokaryote or eukaryote. Additionally, gRNA-SeqRET automates the bulk extraction of any regions of sequence relative to genes or other features, aiding in the design of homology arms for insertion or deletion constructs. We also assess in silico the application of a designed gRNA library to other closely related genomes and demonstrate that for very closely related organisms Average Nucleotide Identity (ANI) > 95% a large fraction of the library may be of relevance. The gRNA-SeqRET web application pipeline can be accessed at https://grna.jgi.doe.gov. The source code is comprised of freely available software tools and customized Python scripts, and is available at https://bitbucket.org/berkeleylab/grnadesigner/src/master/ under a modified BSD open-source license (https://bitbucket.org/berkeleylab/grnadesigner).

Abstract P2177: Autophagy-based Drugs Can Be Used To Treat Anthracycline-induced Cardiotoxicity

Background: Anthracyclines are highly effective chemotherapeutics, but their use is limited by Anthracycline-Induced Cardiotoxicity (AIC) with notable decline in ejection fraction (EF). Recovery of cardiac function is slow and often only partial, mandating novel and more efficacious treatment approaches to AIC. Hypothesis: Based on high throughput genetic screening in zebrafish we identified autophagy activation, e.g. via Atg7, a key protein in the induction of autophagy, as a potential novel translational therapeutic approach to AIC. Methods: In a mouse model of cardiotoxicity (4 x weekly 5 mg/kg intravenous doxorubicin treatment) two approaches were tested starting one week after the last treatment: 1) AdenoAssociated Viral vector 9-mediated overexpression of Atg7 in the myocardium (AAV9-ATG7, one time injection) and 2) 3 FDA approved autophagy activating (FAA) drugs (pravastatin (40 mg/kg), spironolactone (20 mg/kg) and empagliflozin (10 mg/kg), by daily gavage). Echocardiograms were performed at baseline, before start of therapy and after 28 days before sacrifice. Results: All three FAA drugs were able to rescue AIC and Atg7 overexpression by AAV9 alone sufficed to rescue from AIC (Figure 1). All three FAA drugs were able to either restore Atg7 levels to baseline (pravastatin and empagliflozin) or 2.5 times above baseline levels (spironolactone) versus more than 60fold above baseline by an AAV9-based approach. Conclusion: Atg7 overexpression and autophagy activation suffices to rescue from AIC and can be recapitulated to different levels by pravastatin, empaglifozin and spironolactone.

Theory for High-Throughput Genetic Interaction Screening.

Systematic, genome-scale genetic screens have been instrumental for elucidating genotype-phenotype relationships, but approaches for probing genetic interactions have been limited to at most ∼100 pre-selected gene combinations in mammalian cells. Here, we introduce a theory for high-throughput genetic interaction screens. The theory extends our recently developed Multiplexing using Spectral Imaging and Combinatorics (MuSIC) approach to propose ∼105 spectrally unique, genetically encoded MuSIC barcodes from 18 currently available fluorescent proteins. Simulation studies based on constraints imposed by spectral flow cytometry equipment suggest that genetic interaction screens at the human genome-scale may be possible if MuSIC barcodes can be paired to guide RNAs. While experimental testing of this theory awaits, it offers transformative potential for genetic perturbation technology and knowledge of genetic function. More broadly, the availability of a genome-scale spectral barcode library for non-destructive identification of single cells could find more widespread applications such as traditional genetic screening and high-dimensional lineage tracing.

High-throughput functional screen identifies YWHAZ as a key regulator of pancreatic cancer metastasis

Pancreatic cancer is a leading cause of cancer death due to its early metastasis and limited response to the current therapies. Metastasis is a complicated multistep process, which is determined by complex genetic alterations. Despite the identification of many metastasis-related genes, distinguishing the drivers from numerous passengers and establishing the causality in cancer pathophysiology remains challenging. Here, we established a high-throughput and piggyBac transposon-based genetic screening platform, which enables either reduced or increased expression of chromosomal genes near the incorporation site of the gene search vector cassette that contains a doxycycline-regulated promoter. Using this strategy, we identified YWHAZ as a key regulator of pancreatic cancer metastasis. We demonstrated that functional activation of Ywhaz by the gene search vector led to enhanced metastatic capability in mouse pancreatic cancer cells. The metastasis-promoting role of YWHAZ was further validated in human pancreatic cancer cells. Overexpression of YWHAZ resulted in more aggressive metastatic phenotypes in vitro and a shorter survival rate in vivo by modulating epithelial-to-mesenchymal transition. Hence, our study established a high-throughput screening method to investigate the functional relevance of novel genes and validated YWHAZ as a key regulator of pancreatic cancer metastasis.

Abstract 3471: A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment

Abstract Tumor-associated macrophages (TAMs) are important players to maintain immunosuppression within the tumor microenvironment (TME) and attenuate the function of effector immune cells to promote tumor survival. High density of TAMs is well-recognized as a feature of tumor progression and poor prognostic factor across various tumor types. Therefore, therapeutic strategies to dampen TAMs’ suppressive potential in TME and reprogram them towards a pro-inflammatory phenotype are being increasingly appreciated as being paramount for an effective immunotherapy. Nevertheless, the key mediators of TAM-induced immunosuppression remain largely unknown, identification of which can lead to the development of novel immunotherapeutic strategies aimed at re-educating the immunosuppressive TAMs to an anti-tumor phenotype. To systematically interrogate the genes responsible for the immune-suppressive phenotype of TAMs, we developed a functional, high-throughput genetic screening platform (iOTarg™) based on human primary monocyte-derived M2-like macrophages. Using CRISPR, we knocked out a proprietary library of well-expressed and druggable genes (1400 genes) in two independent donor-derived M2-like macrophages and cocultured them with activated autologous effector T cells (TCs). We achieved nearly complete knockout (KO) efficacy over a range of control genes. Functional impact of individual KO on macrophage viability and phenotype, as well as on TC activity, was measured using multiparametric assay readouts. Initial hits were reconfirmed in three additional donors in a secondary screen that integrated an additional tumor lysis readout. As expected, knockout of CSF1R, which is essential for macrophage maintenance, resulted in a dramatic loss of macrophage cell viability, whereas KO of established TAM markers, TREM2 and Clever-1, induced a change in macrophage phenotype. Furthermore, KO of immune-inhibitory receptor LILRB2 reduced the M2-like phenotype and restored TC activity. Inhibition of M2 activation of macrophages and subsequent increase in TC activation culminated in strong tumor cell killing for a subset of genes, highlighting an untapped repertoire of novel TAM-associated immune-checkpoint targets. Taken together, we report for the first time a highly sophisticated target discovery platform that addresses the functional role of any TAM-expressed gene in regulating macrophage viability, phenotype, T cell activity and even its gross impact on tumor cell lysis, all done in a high-throughput format employing CRISPR-edited primary human immune cells and multi-parametric functional immunological assays. As a result, we could confirm well-known targets in clinical testing, as well as identify additional novel targets that could lead to first-in-class, TME-based therapeutics and expansion of treatment options in immune oncology. Citation Format: Kritika Sudan, Tillmann Michels, Carmen Amerhauser, Claudia Tschulik, Leonie Majunke, Lucille Albert, Valentina Volpin, Adriana Turqueti-Neves, Ronny Milde, Nisit Khandelwal. A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3471.

HAP1, a new revolutionary cell model for gene editing using CRISPR-Cas9.

The use of next-generation sequencing (NGS) technologies has been instrumental in the characterization of the mutational landscape of complex human diseases like cancer. But despite the enormous rise in the identification of disease candidate genetic variants, their functionality is yet to be fully elucidated in order to have a clear implication in patient care. Haploid human cell models have become the tool of choice for functional gene studies, since they only contain one copy of the genome and can therefore show the unmasked phenotype of genetic variants. Over the past few years, the human near-haploid cell line HAP1 has widely been consolidated as one of the favorite cell line models for functional genetic studies. Its rapid turnover coupled with the fact that only one allele needs to be modified in order to express the subsequent desired phenotype has made this human cell line a valuable tool for gene editing by CRISPR-Cas9 technologies. This review examines the recent uses of the HAP1 cell line model in functional genetic studies and high-throughput genetic screens using the CRISPR-Cas9 system. It covers its use in an attempt to develop new and relevant disease models to further elucidate gene function, and create new ways to understand the genetic basis of human diseases. We will cover the advantages and potential of the use of CRISPR-Cas9 technology on HAP1 to easily and efficiently study the functional interpretation of gene function and human single-nucleotide genetic variants of unknown significance identified through NGS technologies, and its implications for changes in clinical practice and patient care.