Mutant RNA Research Articles

The Role of Mutant RNA in the Pathogenesis of Huntington’s Disease and Other Polyglutamine Diseases

The Role of Mutant RNA in the Pathogenesis of Huntington’s Disease and Other Polyglutamine Diseases

Off-target effects of cytidine base editor and adenine base editor: What can we do?

Abstract Base editors (BEs) is a powerful tool that could edit the genome efficiently without inducing DNA double-strand breaks (DSBs). Recently, off-target DNA and RNA mutations of BEs were found, rasing concerns about its precision and safety. Here we discuss some strategies that might eliminate off-target effects of BEs.

Solid-Phase Hybridization Assay for Detection of Mutated Cancer DNA by Fluorescence.

We report a straightforward protocol for the detection of mutated DNA extracted fromcancer cells. The assay combines a step-wisesolid-phase hybridization and a readout by fluorescence emission. We detect a single-nucleotide polymorphism in two human oncogenes, BRAF and EGFR, and reach alimit of the detection of 300pM by conventional fluorometry. The protocol described herein may be used as a foundation for development of automatic optimized assays capable for detection of mutant DNA and RNA in vitroand in cells.

The genetic script of metastasis.

Metastasis is a pivotal event that changes the course of cancers from benign and treatable to malignant and difficult to treat, resulting in the demise of patients. Understanding the genetic control of metastasis is thus crucial to develop efficient and sustainable targeted therapies. Here we discuss the alterations in epigenetic mechanisms, transcription, chromosomal instability, chromosome imprinting, non-coding RNAs, coding RNAs, mutant RNAs, enhancers, G-quadruplexes, and copy number variation to dissect the genetic control of metastasis. We conclude that the genetic control of metastasis is predominantly executed through epithelial to mesenchymal transition and evasion of cell death. We discuss how genetic regulatory mechanisms can be harnessed for therapeutic purposes to achieve sustainable control over cancer metastasis.

Reengineering a tRNA Methyltransferase To Covalently Capture New RNA Substrates.

Covalent RNA modifications can alter the function and metabolism of RNA transcripts. Altering the RNA substrate specificities of the enzymes that install these modifications can provide powerful tools to study and manipulate RNA. To develop new tools and probe the plasticity of the substrate specificity of one of these enzymes, we examined the engineerability of the uridine-54 tRNA methyltransferase, TrmA. Starting from a mutant that remains covalently bound to its substrate RNA (E358Q, TrmA*), we were able to use both rational design and a high-throughput sequencing assay to examine the RNA substrates of TrmA*. Although rational engineering substantially changed TrmA* specificity, the rationally designed substrate mutants we developed still retained activity with the wild-type protein. Using high-throughput substrate screening of additional TrmA* mutants, we identified a triple mutant of the substrate RNA (C56A;A58G;C60U) that is efficiently trapped by a TrmA* double mutant (E49R;R51E) but not by the wild-type TrmA*. This work establishes a foundation for using protein engineering to reconfigure substrate specificities of RNA-modifying enzymes and covalently trap RNAs with engineered proteins.

Transcriptional Fidelity of Mitochondrial RNA Polymerase RpoTm from Arabidopsis thaliana

Fidelity of RNA synthesis is essential for the faithful transfer of information from DNA to RNA. A comprehensive analysis of the nucleotide selectivity by the mitochondrial RNA polymerase (RNAP) RpoTm, from Arabidopsis thaliana, has been carried out. The kinetic parameters for the incorporation of cognate, noncognate, and oxidized bases have been determined. The results establish high fidelity of mitochondrial transcription resembling those of replicative polymerases in the absence of repair. In addition, RpoTm incorporates oxidized nucleotides with similar efficiency compared with mismatches and is capable of extending the RNA beyond the insertion of the oxidized base. Furthermore, lesion bypass study on RpoTm demonstrates that the enzyme bypasses 8-oxo-guanine by insertion of adenine leading to C to A mutations in RNA. Homology modeling of RpoTm elongation complex allows delineation of the residues necessary for stabilizing the incoming NTP substrate and for posing the template nucleotide residue. Substitution of these residues leads to compromise in the activity of the enzyme corroborating their importance in RNA synthesis. Comparison of the data with T7 RNAPs indicates that low efficiency of misincorporation is a universal strategy used by single-subunit RNAPs for maintaining high fidelity in the absence of proofreading and repair activity in mitochondria.

Mitochondrial outer membrane voltage-dependent anion channel is involved in renal dysfunction in a spontaneously hypertensive rat carrying transfer RNA mutations

Mitochondrial DNA mutations promote hypertensive renal dysfunction, but the molecular mechanism remains unclear. This study compared renal damage between spontaneously hypertensive rats (SHR) and SHR with mitochondrial transfer (t)RNA mutations. To investigate the role of mitochondrial outer membrane voltage-dependent anion channel 1 (VDAC1) in the process of tRNA-promoting mitochondrial dysfunction, we treated HK-2 cells with H2O2, cyclosporine (CsA), or atractylodin (Atr) to observe the association between VDAC1 and mitochondrial function. Intriguingly, the mitochondrial structure of SHR carrying tRNA mutations was obviously disordered, and reactive oxygen species production and VDAC1 and Bax expression and binding were increased, which was associated with marked renal dysfunction. The expression of VDAC1 and Bax was also up-regulated in HK-2 cells by H2O2 treatment. However, CsA and Atr had no significant effect on the expression of VDAC1 and Bax. H2O2 caused mitochondrial membrane potential collapse, while CsA could increase the mitochondrial membrane potential and Atr had the opposite effect. Treatment with H2O2 significantly decreased ATP synthesis, which was improved by intervention with Atr. H2O2 also decreased the maximum oxygen consumption rate, while CsA and Atr had no significant effect. We found that H2O2 promoted the colocalization of VDAC1 and Bax, which was partially inhibited by intervention with CsA or Atr. In conclusion, we found that tRNA mutations promoted hypertensive renal insufficiency. Increased reactive oxygen species was an important associated mechanism, which inhibited mitochondrial function by affecting VDAC1 expression and function.

P1.03-20 Exploring Driver Mutations in Non-Coding RNAs in Lung Adenocarcinoma

P1.03-20 Exploring Driver Mutations in Non-Coding RNAs in Lung Adenocarcinoma

944P - Evaluation of different diagnostic methods for identification of FGFR alteration in advanced urothelial carcinomas: Proficiency results based on multiple RNA extraction kits and mutation detection methods

BackgroundPan FGFR inhibitors such as Erdafitinib are approved in patients with advanced urothelial carcinoma (aUC) harboring FGFR alterations. Based on the necessity of sensitive and reproducible identification of these alterations in diagnostic routines this study aimed to compare different RNA isolation techniques and the QIAGEN therascreen® FGFR RGQ RT-PCR Kit with the SNaPshot mutational analysis. MethodsNucleic acids were extracted from 47 UC patients using the QIAGEN RNeasy® DSP FFPE Kit, STRATIFYER Xtract or Maxwell RNA (Promega) isolation kit. Mutations of the FGFR3 gene were detected using the SNaPshot method as well as via the QIAGEN therascreen® FGFR RGQ RT-PCR Kit. Intrinsic molecular subtypes were assessed using immunohistochemistry (IHC) of GATA3, FOXA1, CK20, CK5 and CD44 as well as RT-qPCR for gene expression of CK20 and CK5. ResultsAll three different RNA isolation kits showed comparable amount of extracted RNA (Median 290, 177 and 226ng/ul). Six hotspot FGFR3 mutations were identified with 100% concordance. The main difference were the necessary working hours: per 10 samples, 4 vs. 4.5 vs 8hours were needed for Stratifyer, Maxwell or QIAGEN RNA isolation. In addition, of 47 analyzed samples 100% concordance between QIAGEN therascreen® FGFR RGQ RT-PCR Kit and SNapShot analysis were achieved with 18 mutations identified (14 S249C, 2 R248C and 2 Y375). Immunohistochemical basal marker profile (CD44 and CK5) as well as high CK5 expression was observed in 28 (6/28 FGFR3-altered). Luminal marker profile (IHC markers: GATA3, FOXA1 and CK20 as well as expression of CK20) was detected in 37 samples (5/37 with FGFR3 alteration, n.s.). ConclusionsDifferent RNA extraction methods presented with comparable amount of nucleic acids with variant working hours. Moreover, a 100% concordance between the well established SNaPshot analysis compared to the QIAGEN therascreen® FGFR RGQ RT-PCR Kit for FGFR3 mutations was observed. So far, the frequency of FGFR3 mutations in aUC was not related to the type of UC as defined by marker profils. Legal entity responsible for the studyBRIDGE consortium. FundingJanssen. DisclosureR. Wirtz: Officer / Board of Directors: Stratifyer. M. Eckstein: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Janssen. A. Hartmann: Advisory / Consultancy: Janssen. All other authors have declared no conflicts of interest.

Worming into the Uncharacterized Human Proteome.

Using neXtProt release 2019-01-11, we manually curated a list of 1837 functionally uncharacterized human proteins. Using OrthoList 2, we found that 270 of them have homologues in Caenorhabditis elegans, including 60 with a one-to-one orthology relationship. According to annotations extracted from WormBase, the vast majority of these 60 worm genes have RNAi experimental data or mutant alleles, but manual inspection shows that only 15% have phenotypes that could be interpreted in terms of a specific function. One third of the worm orthologs have protein-protein interaction data, and two of these interactions are conserved in humans. The combination of phenotypic, protein-protein interaction, and gene expression data provides functional hypotheses for 8 uncharacterized human proteins. Experimental validation in human or orthologs is necessary before they can be considered for annotation.

Detection of POLE Subtypes in High-Grade Endometrioid Carcinoma by BaseScope-ISH Assay.

Objective: The identification of DNA polymerase epsilon (POLE) mutation subtypes in endometrial cancer is critical for molecular classification. The mutation of the POLE gene could only be detected by sequencing until now. We propose to validate and develop the feasibility of using BaseScope, an in situ hybridization (ISH) assay, for the detection of POLE mutations in high-grade endometrioid carcinomas (EC).Methods: Among 51 paraffin-embedded samples of high-grade EC, BaseScope-ISH assays were used to detect the RNA mutation status of the POLE gene, mainly focusing on two hotspot mutations of P286R and V411L. The number of positive signals in the cytoplasm was counted, setting the positive threshold and determining the in situ hybridization results. The sensitivity and specificity of BaseScope-ISH assay were compared with that of the Sanger sequencing results.Results: Based on the BaseScope assay, there were 19 positive samples and 32 negative samples in a total of 51 samples. Of the 19 positive samples, 10 samples showed P286R site mutations in the POLE gene, while the other nine samples were V411L site mutations. Only one sample with the V411L site mutation identified by Sanger sequencing showed negative signal value. The remaining 31 cases without the P286R site mutation or V411L site mutations all showed negative signal. This analysis result showed the sensitivity was 95% and the specificity was 100% for the BaseScope assay detecting POLE mutants in high-grade EC.Conclusion: In the case of high-grade EC, combined with morphological characteristics, the BaseScope assay can effectively and specifically identify POLE mutation cases, providing a reliable foundation for the application of clinical diagnosis and molecular classification.

Comprehensive Characterization of Somatic Mutations Impacting lncRNA Expression for Pan-Cancer

Somatic mutations have long been recognized as an important feature of cancer. However, analysis of somatic mutations, to date, has focused almost entirely on the protein coding regions of the genome. The potential roles of somatic mutations in human long noncoding RNAs (lncRNAs) are therefore largely unknown, particularly their functional significance across different cancer types. In this study, we characterized some lncRNAs whose expression was affected by somatic mutations (defined as MutLncs) and constructed global MutLnc landscapes across 17 cancer types by systematically integrating multiple levels of data. MutLncs were commonly downregulated and carried low mutation frequencies and non-silent mutations in most cancer types. Co-occurrence analysis in pan-cancer highlighted combined patterns of specific MutLncs, suggesting that a number of MutLncs influence diverse cancer types through combination effects. Several conserved and cancer-specific functions of MutLncs were determined. We further explored the somatic mutations affecting lncRNA expression via mixed and unmixed effects, which led to specific functions in pan-cancer. Survival analysis indicated that MutLncs and co-occurrence pairs can potentially serve as cancer biomarkers. Clarification of the specific roles of MutLncs in human cancers could be beneficial for understanding the molecular pathogenesis of different cancer types and developing the appropriate treatments.

English

The reaction of four wheat RbcS RNAi mutants and wild type to the infection of M. incognita was investigated in a greenhouse pot experiment using plant growth parameters as well as nematode reproduction. Obtained results revealed that all estimated growth parameters of lines under study were reduced by M. incognita infection to various extents. RSS13.2 line showed the highest percentage of reduction of plant total fresh weight, length, and shoot dry weight. It did act as good host for root-knot nematode; therefore, it was classified as susceptible host. In addition, the lowest reduction percentage of fresh and dry weights was observed in RSS8.5 line although it showed high values for nematode development and reproduction parameters; therefore, it seemed to be as a tolerant host. The maximum reduction in chlorophyll content was recorded in RSS7.6 line, whereas, the minimum was obtained from RSS11.5 line. It was evident that none of the five tested wheat lines was immune to M. incognita infection. The lowest numbers of second stage juveniles, galls, and egg-masses were observed in soil and on roots of RSS11.5 line although its plant growth was slightly affected; therefore, it is considered as partially resistant line. It could be concluded from the results of this study that, RNAi mutants of the RbcS were partially effective in suppressing root-knot nematode development. © 2019 Friends Science Publishers

Mutations in the Helicobacter pylori 23S rRNA gene associated with clarithromycin resistance in patients at an endoscopy unit in Medellín, Colombia

Introduction: Clarithromycin is the first-line antibiotic for the treatment of Helicobacter pylori infection. Bacterial resistance is mainly due to the presence of specific mutations in the 23S ribosomal RNA (rRNA) gene. Objective: To determine the frequency of A2143G and A2142G specific mutations in the 23S rRNA gene associated with clarithromycin resistance of H. pylori in samples from patients with dyspeptic manifestations in Medellín, northwestern Colombia. Materials and methods: DNA was extracted from gastric biopsy samples of patients with dyspeptic manifestations seen at an endoscopy unit in Medellín between 2016 and 2017. PCR was performed to amplify the bacterial s and m vacA regions, and a region in the 23S rRNA gene. The presence of the A2142G and A2143G mutations was determined using the restriction fragment length polymorphism (RFLP) technique with the BbsI and BsaI enzymes, respectively. Results: The prevalence of infection was 44.2% (175/396), according to the histopathology report. The positive samples were analyzed and the three regions of the bacterial genome were amplified in 143 of the 175 samples. The A2143G and A2142G mutations were identified in 27 samples (18.8%, 27/143). The most frequent mutation was A2143G (81.5%, 22/27). Conclusions: We found a high prevalence of H. pylori mutations associated with clarithromycin resistance in the study population. Further studies are required to determine the bacterial resistance in the Colombian population in order to define first line and rescue treatments.

Identifying the growth associating genes of Nannochloropsis oceanica by bulked mutant analysis (BMA) and RNA sequencing (BMR-seq)

Microalgal strains (lines), either economically or ecologically important or both, are usually clones derived from a single cell. The functions of their genes are verified usually through either loss of function or gain of function approaches, for example, overexpression, knockout, silencing, and editing. In comparison with plants and animals, the genetic studies of microalgae are scarce, and less genes were de novo cloned. Bulked segregant analysis (BSA) and bulked segregant RNA sequencing (BSR-seq) are widely used to identify the linking markers or control genes of sexually reproducing organisms. In order to avoid genotyping, a large amount of individuals in genome-wide association study (GWAS), we modified BSA and BSR-seq in this study to bulked mutant analysis (BMR) and bulked mutant RNA sequencing (BMR-seq). In order to generate stable variations, we used Zeocin to cause the double-strand breaks of DNA (DSBs), and generate rich InDel variations during DNA repairing. We identified a set of growth performance associating SNPs and InDels among the bulked DNA and bulked RNA. We found that the function-known genes either containing these variations or locating upstream or downstream of these variations are highly related to the complex growth performance trait of Nannochloropsis oceanica. Our findings verified that Zeocin is effective to create rich InDels in monoploid and asexual N. oceanica genome, and BMA and BMR-seq are applicable for identifying the associating SNPs and InDels of the growth performance trait. We prefer to use BMA to identify trait associating genes and variations; microalgae are fast growing with a short generation time, and mRNAs are the transcripts of genes and share the variations with DNA. We believe that the methods we developed in this study will facilitate the genetic studies of the traits of diverse microalgae.

Engineering Corynebacterium glutamicum for high-titer biosynthesis of hyaluronic acid

Hyaluronic acid (HA) is a member of the glycosaminoglycan family and has been widely used in the clinical, medical, cosmetic and food industries. In this study, we constructed a superior cell factory in Corynebacterium glutamicum for high-titer HA biosynthesis through systematic design and metabolic engineering based on a genome-scale metabolic model, iCW773. The OptForceMUST algorithm was used in iCW773 to determine genetic interventions by using flux balance analysis. Enhancement of the HA biosynthesis pathway and attenuation of the glycolysis pathway, the pentose phosphate pathway and the dehydrogenation of pyruvate were predicted as targets for genetic modulations. Various genetic strategies were employed, including an additional promoter, PdapB, driving hasB expression, antisense RNA-mediated attenuation of fba, zwf deletion and lactate/acetate pathway knockout. The integrated genetic changes in recombinant C. glutamicum produced 24.5 g/L HA in a fed-batch culture. Finally, pyruvate dehydrogenase activity was further reduced by antisense RNA and initial codon mutation to divert carbon flux from byproducts to HA. The corresponding modified strain, CgHA25, achieved a titer of 28.7 g/L. The byproduct concentration was reduced by half, and the major weight-average molecular weight (Mw) component was 0.21 MDa. This work reports a significant improvement in the HA titer in a safe host achieved by systematic metabolic engineering.

Antibody-Free Assay for RNA Methyltransferase Activity Analysis.

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Impact of 5′-end nucleotide modifications of HIV-1 genomic RNA on reverse transcription

Reverse transcription of retroviral RNA is accomplished through a minus-strand strong stop cDNA (-sscDNA) synthesis and subsequent strand-transfer reactions. We have previously reported a critical role of guanosine (G) number at 5′-terminal of HIV-1 RNA for successful strand-transfer of -sscDNA. In this study, role(s) of the cap consisting of 7-methyl guanosine (7mG), a hallmark of transcripts generated by RNA polymerase II, at the 5′-end G nucleotide (5′-G) of HIV-1 RNA were examined. In parallel, contribution of highly conserved GGG tract located at the U3/R boundary in 3′ terminal region of viral RNA (3′-GGG tract) was also addressed. The in vitro reverse transcription analysis using synthetic HIV-1 RNAs possessing the 5′-G with cap or triphosphate form demonstrated that the 5′-cap significantly increased strand-transfer efficiency of -sscDNA. Meanwhile, effect of the 5′-cap on the strand-transfer was retained in the reaction using mutant HIV-1 RNAs in which two Gs were deleted from the 3′-GGG tract. Lack of apparent contribution of the 3′-GGG tract during strand-transfer events in vitro was reproduced in the context of HIV-1 replication within cells. Instead, we noticed that the 3′-GGG tract might be required for efficient gene expression from proviral DNA. These results indicated that 7mG of the cap on HIV-1 RNA might not be reverse-transcribed and a possible role of the 3′-GGG tract to accept the non-template nucleotide addition during -sscDNA synthesis might be less likely. The 5′-G modifications of HIV-1 RNAs by the cap- or phosphate-removal enzyme revealed that the cap or monophosphate form of the 5′-G was preferred for the 1st strand-transfer compared to the triphosphate or non-phosphate form. Taken together, a status of the 5′-G determined strand-transfer efficiency of -sscDNA without affecting the non-template nucleotide addition, probably by affecting association of the 5′-G with 3′-end region of viral RNA.

Abstract 5248: Expression of somatic alleles in TCGA data reveals allelic imbalance with implications for treatment and survival

Abstract Next-generation technologies, including whole-genome sequencing of DNA and RNA, have facilitated the identification of different cancer subclasses and genes responsible for tumor growth and progression, and guide the application of targeted therapies for personalized medicine. In this study, we employ a novel, combined approach for assessing cancer mutations. As standard practice, our group inspects individual DNA sequencing reads underlying each somatic mutation call to assess quality and confirm calls made by variant calling algorithms. This approach provides confidence in identifying likely candidate driver genes while eliminating calls unsupported by aligned reads. We have recently expanded this method to include the assessment of RNA variants as well. In this approach, called DNA mutations are inspected in sequencing reads from both DNA and RNA. In analysis of 850 tumors from the TCGA breast cancer dataset, we observed inconsistent levels of mutation expression i.e., RNA imbalance, where only less than half of the mutations identified have similar variant call ratios in both RNA and DNA. We observe three types of RNA allelic imbalance: higher RNA mutation call ratios compared to that in DNA, higher DNA mutation call ratios compared to that in RNA, and RNA expression where the DNA mutation is absent. We also observe sites where a mutation is present in DNA, but the RNA is not expressed. Notably, we found a patient with a E545K mutation in PIK3CA DNA that was not expressed in the RNA, suggesting that treatment with PI3 kinase inhibitors will be ineffective for this patient. When analyzing the most commonly mutated genes in this cohort, we found that the mutation expression patterns are not random but specific to individual genes. For example, GATA3 mutations are almost always expressed with a higher RNA call ratio, BRCA1 and LRP6 mutations are rarely expressed, and PIK3CA mutations are almost always expressed with the same call ratio. We also noticed differences in survival patterns with the different categories of RNA expression for certain genes. We found that patients with higher RNA call ratios in GATA3 have better overall survival compared with patients that have lower RNA call ratios, which is consistent with reported better survival outcome for patients with GATA3 mutations. We also observed that patients with lower RNA mutation call ratios in TP53 had better survival, and those with higher MAP3K1 call ratios had better overall survival. As DNA and RNA are commonly assayed in cancer studies such as TCGA, we present a novel, combined approach for better identifying mutations of interest. We believe this approach provides an improved methodology for assessing mutations in patients and providing insight into patient outcome. These results also suggest caution when evaluating DNA alone and the necessity for confirming the expression of mutations in RNA. Citation Format: Shannon T. Bailey, Jim Lund, Jeffrey R. Gulcher. Expression of somatic alleles in TCGA data reveals allelic imbalance with implications for treatment and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5248.

Abstract 2584: Exosome molecular reference standards (Exo-Ref) generated from CRISPR/Cas9 engineered cell Lines for oncology diagnostic applications

Abstract Introduction: Exosomes (commonly known as extracellular vesicles) have generated tremendous interest for the therapeutic and diagnostic applications for oncology and other diseases. Specifically, exosome molecular cargo (proteins, nucleic acid, and small molecules) profiling have been subject of intense research for potential biomarkers for cancer. Currently, there exist only few exosomal nucleic acid (ExoRNA) biomarkers that have been realized for cancer diagnostics and treatment monitoring. This is due to the lack of exosome reference standards for assay development, assay performance validation, and interpretation of results. Exo-Ref products are ideal reference standards, since they mimic physical properties and genomic composition of exosomes isolated from patient biofluids. Further, Exo-Ref are biologically relevant, reproducible, and uses engineered cell lines that have previously demonstrated a source of reference materials (ctDNA, FFPE, and cells). We have developed Exo-Ref featuring RNA SNPs including point mutations in EGFR (c.2369C>T, c.2573T>G), NRAS (c.181C>A), and PIK3A(c.1633G>A). Herein we present the isolation of exosomes from engineered cell lines and characterization of exo-RNA SNPs transcripts using RT-digital PCR and Next Generation Sequencing (NGS). The validation of variants using the two methods show a great promise for Exo-Ref products for oncology exosome-based diagnostic applications. Methods: CRISPR/Cas9 targeting reagents were transfected into either HCT116 or RKO cell line. Exosomes are produced by culturing the cells in exosome free culture media. Exosomes were then isolated from ExoRNeasy kit (Qiagen). For genetic analysis, exo-RNA was isolated from using trizol/membrane filter using the ExoRNeasy kit and allelic rare mutations in RNA were verified by RT dPCR and validated by NGS. Results: Engineered, homozygous clones were conclusively identified by Sanger Sequencing. We were able to reproducibly achieve fragmentation profile of exo-RNA centered at approximately 25bp. Digital RT-PCR validated cellular and exosome mutant transcripts. Targeted variants including EGFR-T790M, EGFR-L858R, PIK3CA-E45K, NRAS-Q61K showed measurable copies of EV-RNA and cell-RNA from engineered cells. Digital PCR verified variants in exo-RNA was also detected by NGS and were consistent with dPCR. NGS sequence validation of exo-RNA transcript were verified at 100% mutation frequency. Conclusion CRISPR/Cas9 engineered cell lines are biologically relevant materials and allow for a reproducible and renewable source of Exo-RNA reference standards for assay development and quality control for use in clinical exosome-based diagnostic workflows. Citation Format: Vigneshwaran Mani, Suhani Thakker, Debapriya Sengupta, Sravanthi Kondapalli, Gianluca Roma, Qi Zheng, Sonika Saddar. Exosome molecular reference standards (Exo-Ref) generated from CRISPR/Cas9 engineered cell Lines for oncology diagnostic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2584.