Georgia Giannoukos

We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.

Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.

Targeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that approximately 60% of target bases in the exonic 'catch', and approximately 80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

Although the composition of the human microbiome is now well-studied, the microbiota's >8 million genes and their regulation remain largely uncharacterized. This knowledge gap is in part because of the difficulty of acquiring large numbers of samples amenable to functional studies of the microbiota. We conducted what is, to our knowledge, one of the first human microbiome studies in a well-phenotyped prospective cohort incorporating taxonomic, metagenomic, and metatranscriptomic profiling at multiple body sites using self-collected samples. Stool and saliva were provided by eight healthy subjects, with the former preserved by three different methods (freezing, ethanol, and RNAlater) to validate self-collection. Within-subject microbial species, gene, and transcript abundances were highly concordant across sampling methods, with only a small fraction of transcripts (<5%) displaying between-method variation. Next, we investigated relationships between the oral and gut microbial communities, identifying a subset of abundant oral microbes that routinely survive transit to the gut, but with minimal transcriptional activity there. Finally, systematic comparison of the gut metagenome and metatranscriptome revealed that a substantial fraction (41%) of microbial transcripts were not differentially regulated relative to their genomic abundances. Of the remainder, consistently underexpressed pathways included sporulation and amino acid biosynthesis, whereas up-regulated pathways included ribosome biogenesis and methanogenesis. Across subjects, metatranscriptional profiles were significantly more individualized than DNA-level functional profiles, but less variable than microbial composition, indicative of subject-specific whole-community regulation. The results thus detail relationships between community genomic potential and gene expression in the gut, and establish the feasibility of metatranscriptomic investigations in subject-collected and shipped samples.

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis.

Promising new sequencing technologies, based on sequencing-by-synthesis (SBS), are starting to deliver large amounts of DNA sequence at very low cost. Polymorphism detection is a key application. We describe general methods for improved quality scores and accurate automated polymorphism detection, and apply them to data from the Roche (454) Genome Sequencer 20. We assess our methods using known-truth data sets, which is critical to the validity of the assessments. We developed informative, base-by-base error predictors for this sequencer and used a variant of the phred binning algorithm to combine them into a single empirically derived quality score. These quality scores are more useful than those produced by the system software: They both better predict actual error rates and identify many more high-quality bases. We developed a SNP detection method, with variants for low coverage, high coverage, and PCR amplicon applications, and evaluated it on known-truth data sets. We demonstrate good specificity in single reads, and excellent specificity (no false positives in 215 kb of genome) in high-coverage data.

Background Pediatric inflammatory bowel disease (IBD) is challenging to diagnose because of the non-specificity of symptoms; an unequivocal diagnosis can only be made using colonoscopy, which clinicians are reluctant to recommend for children. Diagnosis of pediatric IBD is therefore frequently delayed, leading to inappropriate treatment plans and poor outcomes. We investigated the use of 16S rRNA sequencing of fecal samples and new analytical methods to assess differences in the microbiota of children with IBD and other gastrointestinal disorders. Methodology/Principal Findings We applied synthetic learning in microbial ecology (SLiME) analysis to 16S sequencing data obtained from i) published surveys of microbiota diversity in IBD and ii) fecal samples from 91 children and young adults who were treated in the gastroenterology program of Children’s Hospital (Boston, USA). The developed method accurately distinguished control samples from those of patients with IBD; the area under the receiver-operating-characteristic curve (AUC) value was 0.83 (corresponding to 80.3% sensitivity and 69.7% specificity at a set threshold). The accuracy was maintained among data sets collected by different sampling and sequencing methods. The method identified taxa associated with disease states and distinguished patients with Crohn’s disease from those with ulcerative colitis with reasonable accuracy. The findings were validated using samples from an additional group of 68 patients; the validation test identified patients with IBD with an AUC value of 0.84 (e.g. 92% sensitivity, 58.5% specificity). Conclusions/Significance Microbiome-based diagnostics can distinguish pediatric patients with IBD from patients with similar symptoms. Although this test can not replace endoscopy and histological examination as diagnostic tools, classification based on microbial diversity is an effective complementary technique for IBD detection in pediatric patients.

RNAtag-seq barcodes transcripts prior to cDNA synthesis, thereby allowing pooled library generation from many input samples. Although RNA-seq is a powerful tool, the considerable time and cost associated with library construction has limited its utilization for various applications. RNAtag-Seq, an approach to generate multiple RNA-seq libraries in a single reaction, lowers time and cost per sample, and it produces data on prokaryotic and eukaryotic samples that are comparable to those generated by traditional strand-specific RNA-seq approaches.

We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA.

Understanding the diversity of repair outcomes after introducing a genomic cut is essential for realizing the therapeutic potential of genomic editing technologies. Targeted PCR amplification combined with Next Generation Sequencing (NGS) or enzymatic digestion, while broadly used in the genome editing field, has critical limitations for detecting and quantifying structural variants such as large deletions (greater than approximately 100 base pairs), inversions, and translocations.To overcome these limitations, we have developed a Uni-Directional Targeted Sequencing methodology, UDiTaS, that is quantitative, removes biases associated with variable-length PCR amplification, and can measure structural changes in addition to small insertion and deletion events (indels), all in a single reaction. We have applied UDiTaS to a variety of samples, including those treated with a clinically relevant pair of S. aureus Cas9 single guide RNAs (sgRNAs) targeting CEP290, and a pair of S. pyogenes Cas9 sgRNAs at T-cell relevant loci. In both cases, we have simultaneously measured small and large edits, including inversions and translocations, exemplifying UDiTaS as a valuable tool for the analysis of genome editing outcomes.UDiTaS is a robust and streamlined sequencing method useful for measuring small indels as well as structural rearrangements, like translocations, in a single reaction. UDiTaS is especially useful for pre-clinical and clinical application of gene editing to measure on- and off-target editing, large and small.

A complex microbiota inhabits various microenvironments of the gut, with some symbiotic bacteria having evolved traits to invade the epithelial mucus layer and reside deep within the intestinal tissue of animals. Whether these distinct bacterial communities across gut biogeographies exhibit divergent behaviours is largely unknown. Global transcriptomic analysis to investigate microbial physiology in specific mucosal niches has been hampered technically by an overabundance of host RNA. Here, we employed hybrid selection RNA sequencing (hsRNA-Seq) to enable detailed spatial transcriptomic profiling of a prominent human commensal as it colonizes the colonic lumen, mucus or epithelial tissue of mice. Compared to conventional RNA-Seq, hsRNA-Seq increased reads mapping to the Bacteroides fragilis genome by 48- and 154-fold in mucus and tissue, respectively, allowing for high-fidelity comparisons across biogeographic sites. Near the epithelium, B. fragilis upregulated numerous genes involved in protein synthesis, indicating that bacteria inhabiting the mucosal niche are metabolically active. Further, a specific sulfatase (BF3086) and glycosyl hydrolase (BF3134) were highly induced in mucus and tissue compared to bacteria in the lumen. In-frame deletion of these genes impaired in vitro growth on mucus as a carbon source, as well as mucosal colonization of mice. Mutants in either B. fragilis gene displayed a fitness defect in competing for colonization against bacterial challenge, revealing the importance of site-specific gene expression for robust host-microbial symbiosis. As a versatile tool, hsRNA-Seq can be deployed to explore the in vivo spatial physiology of numerous bacterial pathogens or commensals.

We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.

For the past 25 years we have used a comparative strategy designed to identify anddescribe the endocrine parameters of the oviparous-vivparous transition and subsequent gradual reduction in hepatic yolk protein precursor (vitellogenin) synthesis associated with placental viviparity. Our approach has been to study vertebrate groups in which both oviparous and viviparous modes are common (reptiles, elasmobranchs). We have provided evidence for the control of follicular (granulosa/theca) and luteal steroidogenesis, and the cellular basis of gonadal steroid hormone action on the key target tissues (oviduct, liver). Our results, some of which are summarized below, have led us to suggest that ovarian progesterone (follicular or luteal in origin) has a dual role in the evolution of viviparity: 1. To inhibit myometrial contractions, thus providing a primary condition for egg retention and viviparity. 2. To inhibit estrogen-induced hepatic vitellogenin synthesis as part of both normal oviparous cycles and as a concomitant of placental evolution.

Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage.To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling.These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

The glucocorticoid receptor (GR) HBD must be bound to the protein chaperone hsp90 in order to acquire the high affinity steroid binding conformation. Despite this crucial role of hsp90, its binding site in GR remains poorly defined. Large portions of the GR HBD have been implicated and no similarity has been established between steroid receptor HBDs and the catalytic domains of the protein kinases (<i>e.g.</i> pp60^src, Raf) that also form stable heterocomplexes with hsp90. Thus, it has been thought that some general property of the proteins, such as exposure of hydrophobic residues in partially denatured regions, determines the assembly of stable hsp90 heterocomplexes. In this work, we have studied fusion proteins containing glutathione <i>S</i>-transferase (GST) and very short amino-terminal truncations just before and at the beginning of the rat GR HBD that are otherwise intact to the carboxyl terminus. Overexpression in COS cells of the chimeras GST537C and GST547C was found to yield receptors that were bound to hsp90 and had wild-type steroid binding affinity. However, removal of 7 more amino acids to form GST554C resulted in a fusion protein that did not bind either hsp90 or steroid. Additional mutations revealed that the role of these 7 amino acids was neither to provide a spacer between protein domains nor to expose a protein surface by introducing a bend in the conserved α-helix. Instead, these observations support a model in which the sequence of the 7 amino acids directly or indirectly affects hsp90 binding to the GR HBD. Thus, a region of GR that has not been thought to be relevant for hsp90 binding is now seen to be of critical importance, and these data argue strongly against the commonly accepted model of receptor-hsp90 heterocomplex assembly in which the chaperone initially interacts nonspecifically with hydrophobic regions of the partially denatured HBD and subsequently assists its folding to the steroid binding confirmation.

We have designed resequencing microarrays to test the performance of this platform when interrogating a large number of exons (164 total) from genes associated with cancer. To evaluate false positive and negative rates, dideoxy sequencing was done for 335,420 bases interrogated by the arrays. From the array data, calls could be made for approximately 97.5% of the bases, and false positive rates were very low with only a single mutation reported from the array dataset for which the corresponding dideoxy trace had a clean wildtype sequence. For the nucleotide positions where array calls were made, false negative rates were 1.41% for heterozygous mutations. All the homozygous mutations were detected, but 8.11% were erroneously reported as heterozygous changes from the reference sequence by the array analysis software. In addition, 20 non-small cell lung cancer (NSCLC) samples were analyzed using the arrays, and both somatic and germline mutations were found. The most interesting findings were two MET mutations that have recently been implemented in NSCLC. Large scale MALDI-TOF genotyping indicated that one of these mutations (T1010I) might represent a true cancer-causing genotype, whereas the other (N375S) appears to be a common germline polymorphism.

Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding.We recently reported that seven amino acids (547-553) overlapping the aminoterminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding.The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in fulllength GR and glutathione S-transferase chimeras.Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding.Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/ L553S) was virtually inactive.This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex.These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg 651 .The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550 -554 acting as a hydrophobic clasp.

A pharmacologically relevant property of steroid hormone-regulated gene induction is the partial agonist activity of antisteroid complexes. We now report that dexamethasone-mesylate (Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of the glucocorticoid-selective steroid dexamethasone (Dex), are two new antiprogestins with significant amounts of agonist activity with both the A and B isoforms of progesterone receptor (PR), for different progesterone-responsive elements, and in several cell lines. These compounds continue to display activity under conditions where another partial antiprogestin (RTI-020) is inactive. These new antiprogestins were used to determine whether the partial agonist activity of PR complexes can be modified by changing concentrations of receptor or coregulator, as we have recently demonstrated for glucocorticoid receptors (GRs). Because GR and coregulator concentrations simultaneously altered the position of the physiologically relevant dose-response curve, and associated EC50, of GR-agonist complexes, we also examined this phenomenon with PR. We find that elevated PR or transcriptional intermediary factor 2 (TIF2) concentrations increase the partial agonist activity of Dex-Mes and Dex-Ox, and the EC50 of agonists, independently of changes in total gene transactivation. Furthermore, the corepressors SMRT (silencing mediator for retinoid and thyroid receptors) and NCoR (nuclear receptor corepressor) each suppresses gene induction but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces the EC50 and increases the partial agonist activity of antiprogestins. These comparable responses of GR and PR suggest that variations in receptor and coregulator concentrations may be a general mechanism for altering the induction properties of other steroid receptors. Finally, the magnitude of coregulator effects on PR induction properties are often not identical for agonists and the new antagonists, suggesting subtle mechanistic differences. These properties of Dex-Mes and Dex-Ox, plus the sensitivity of their activity to cellular differences in PR and coregulator concentrations, make these steroids potential new SPRMs (selective progesterone receptor modulators) that should prove useful as probes of PR induction properties.

Progesterone (PR) and estrogen (ER) receptors were previously identified and characterized in the reproductive tract of the turtle,Chrysemys picta,and changes in PR levels were monitored during the seasonal cycle. To understand the hormonal regulation of PR, intact and ovariectomized animals were treated with estradiol, progesterone, and a combination of estradiol and progesterone, and high affinity PR and ER levels were determined by radioligand binding studies. Ovariectomy significantly decreased ER levels; in contrast, PR levels increased following ovariectomy. In both intact and ovariectomized animals, estradiol alone did not elevate PR levels above control; however, the PR was down-regulated by progesterone. ER levels in ovariectomized animals were not restored by any of the steroid regimens. By Western blot analysis, PR levels appeared to increase following ovariectomy, were unaffected by estradiol, and were somewhat decreased following progesterone treatment in estradiol-primed ovariectomized animals. While not quantitative, these results are supportive of radioligand binding studies. Immunocytochemical studies of oviduct PR followed the same pattern showing increased immunoreactivity following ovariectomy, no change with estradiol, and a decrease following progesterone treatment of estradiol-primed animals. Oviduct contractility was monitored as a physiological index of progesterone action. Estradiol significantly increased the amplitude of the contractions bothin vivoandin vitro,whereas progesterone in combination with estradiol significantly inhibited the estrogen effect. This study suggests that estradiol alone may not be adequate for regulation of both ER and PR. While progesterone down-regulates its own receptor, it does not appear to influence the ER. These data are in contrast to mammalian and avian studies which show that estradiol increases both the ER and PR in the reproductive tract, and progesterone down-regulates both receptors.

Adequate evidence exists to suggest the importance of temporal changes in steroid hormone ratios in the normal reproductive/vitellogenin cycle in oviparous and viviparous elasmobranchs and reptiles. In oviparous species, where the cycle is relatively short, secretion of gonadal hormones is synchronous; thus inhibitory actions of progesterone (P) on hepatic or reproductive tract functions would be offset by stimulatory actions of estradiol (E), resulting in appropriate vitellogenin secretion and reproductive tract development. In viviparous species, temporal asynchrony of E and P secretion occurs, and the actions of the individual hormones can be more easily dissected out. Thus, during gestation, where P is the dominant hormone, antagonistic or stimulatory actions of E may be prevented, and the inhibitory action of P on vitellogenesis dominant. Hence vitellogenesis is limited to the follicular phase and eggs are retained. Although the elasmobranch and reptilian species discussed here do not form a continuum through phylogenesis, but rather are extant forms of a particular line of evolution, it is possible to extrapolate from these observations to the probable endocrine interactions in a species as viviparity evolves from oviparity. The theoretical intermediate stage would involve; (a) egg retention, (b) extension of the luteal phase and increased P secretion and (c) resulting in E/P asynchrony and potential expression of "independent" P action, egg retention and yolk suppression.

In non-mammals, estrogen-induced yolk precursors produced by the adult female liver are the main nutritional source for development. Evidence exists that progesterone exerts counter-regulatory effects on estrogen-induced vitellogenesis, and we have used the turtle model (Chrysemys picta) to study changes in hepatic progesterone receptor during the vitellogenic cycle. Using radioligand methods, we show that high and lower affinity binding sites are present in the cytosolic but not nuclear extracts. The lower affinity site is detectable at all times of the year; the high affinity site is mainly observed during non-vitellogenic periods and does not correlate with plasma estrogen. DNA-cellulose chromatography shows that PR-A is present in spring, summer, and winter, and that PR-B is down-regulated except in animals which recently laid eggs. Western blots confirm the presence of PR in all months, but PR-A (88 kDa) is the dominant isoform. PR-B (125 kDa) is well correlated with the luteal phase, winter and fall. Immunocytochemical studies show that PR is nuclear in location, and nuclear heat shock protein 90 (hsp90) is present. Competitive binding studies of both sites reveal that progesterone is the most effective ligand for both, followed by pregnenolone, deoxycorticosterone, and R5020. RU 486 does not bind to the high affinity site but binds moderately well to the lower affinity site. This study suggests that progesterone receptor isoforms are differentially expressed and may be involved as transcriptional regulators of hepatic function outside the periods of active vitellogenesis in the turtle.

Abstract Beta hemoglobinopathies resulting from dysfunctional or deficient adult beta-globin expression are some of the most prevalent inherited blood disorders in the world. Upregulation of developmentally-silenced fetal gamma-globin would replace adult beta-globin to ameliorate disease symptoms. One of the approaches to reactivate fetal globin expression in erythroid cells is through gene editing by zinc finger or CRISPR-Cas9 nucleases to disrupt the expression of a transcription factor BCL11A, which mediates fetal globin silencing. As BCL11A-deficiency leads to hematopoietic stem cells (HSCs) defects, the current editing approaches target the BCL11A erythroid-enhancer region located in intron 2 of the BCL11A gene to selectively reduce BCL11A expression in erythroid cells. Instead of targeting BCL11A, we sought to identify novel cis-regulatory elements at the beta-globin locus for targeted gene editing to achieve fetal globin reactivation. From a lenti-CRISPR mediated saturated mutagenesis screen covering the beta-globin locus using Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP)-2 cells, multiple fetal hemoglobin (HbF)-inducing genomic domains were identified. Most of the hits were concentrated at the gamma-globin (HBG1/2) promoters, clustered at known hereditary persistence of fetal hemoglobin (HPFH) mutation hotspots. In-depth genotype to phenotype analysis further defined the indels responsible for HbF induction in these subdomains. We interrogated multiple families of nucleases and guide RNA (gRNA) combinations with or without single-stranded oligodeoxynucleotides (ssODN) to guide editing outcome. gRNAs were selected based on their HbF induction potential (up to 40%) when introduced into mobilized peripheral blood (mPB) CD34+ hematopoietic stem and progenitor cells (HSPCs) as ribonucleoprotein (RNP) complexes. HSPCs transfected with RNPs targeting either the BCL11A erythroid-enhancer or the HBG1/2 proximal regions were then injected into NBSGW mice to study the editing in SCID-repopulating cells (SRC) and their multilineage reconstitution potential. All groups achieved high levels of human chimerism (&gt;70% hCD45+/hCD45+mCD45) and comparable monocytes, granulocytes, B lymphocytes, and hCD34+ HSPCs reconstitution. However, BCL11A-edited cells showed selective reduction in erythroid lineage (CD235a+) output, up to 4-fold lower than untreated or HBG1/2 promoter-edited HSPCs. Sequencing analysis from lineage-specific sorted cells further revealed reduced editing levels at BCL11A erythroid-specific enhancer in the erythroid compartment compared to unfractionated bone marrow (BM) or other human lineages (70% in erythroid vs. 90% in all other lineages). Furthermore, the nonproductive fraction of indels that did not disrupt the BCL11A GATAA motif was significantly enriched in erythroid cells (22% in erythroid vs. 8% in other lineages). Ex vivo erythroid cultures suggests BCL11A erythroid-enhancer editing may lead to slightly increased apoptosis during erythroid differentiation. In contrast, HBG1/2 promoter-edited cells had similar editing levels and indel patterns across all lineages with no significant lineage skewing. When chimeric BM from HBG1/2 promoter-edited groups were cultured in erythroid conditions, ex vivo-derived erythroid cells had significantly elevated levels of HbF compared to controls. When CD235a+ cells were sorted without further culture from chimeric BM of mice engrafted with HBG1/2 promoter-edited cells, significantly increased levels of HbF were detected by UPLC (up to 30%) compared to controls (~6%). Thus, long-term HSCs have been edited productively at the HBG1/2 promoters. These cells were able to generate erythroid progenitors that harbor HbF-inducing indels, which in turn, gave rise to erythroid cells in vivo with a clinically-relevant levels of HbF in a xenotransplantation model. Together, our data suggest that BCL11A-edited cells have an erythroid differentiation defect or survival disadvantage in NBSGW mouse model that warrants further investigation. In contrast, editing of the HBG1/2 promoters in mPB CD34+ cells achieved sustained HbF expression in erythroid lineage while maintaining multilineage differentiation potential. Targeting of the HBG1/2 promoters in HSPCs may be an attractive strategy for the development of potential gene editing medicines for beta hemoglobinopathies. Disclosures Chang: Editas Medicine Inc.: Employment, Equity Ownership. Sanchez:Editas Medicine Inc.: Employment, Equity Ownership. Heath:Editas Medicine Inc.: Employment, Equity Ownership. deDreuzy:Editas Medicine Inc.: Employment, Equity Ownership. Haskett:Editas Medicine Inc.: Employment, Equity Ownership. Vogelaar:Editas Medicine Inc.: Employment. Gogi:Editas Medicine Inc.: Employment, Equity Ownership. Da Silva:Editas Medicine Inc.: Employment, Equity Ownership. Wang:Editas Medicine Inc.: Employment, Equity Ownership. Sadowski:Editas Medicine Inc.: Employment, Equity Ownership. Gotta:Editas Medicine Inc.: Employment, Equity Ownership. Siwak:Editas Medicine Inc.: Employment, Equity Ownership. Viswanathan:Editas Medicine Inc.: Employment, Equity Ownership. Loveluck:Editas Medicine Inc.: Employment, Equity Ownership. Chao:Editas Medicine Inc.: Employment, Equity Ownership. Tillotson:Editas Medicine Inc.: Employment, Equity Ownership. Chalishazar:Editas Medicine Inc.: Employment, Equity Ownership. Dass:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Brennan:Editas Medicine Inc.: Employment, Equity Ownership. Tabbaa:Editas Medicine Inc.: Employment, Equity Ownership. Marco:Editas Medicine Inc.: Employment, Equity Ownership. Zuris:Editas Medicine Inc.: Employment, Equity Ownership. Reyon:Editas Medicine Inc.: Employment, Equity Ownership. Isik:Editas Medicine Inc.: Employment, Equity Ownership. Friedland:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Harbinski:Editas Medicine Inc.: Employment, Equity Ownership. Giannoukos:Editas Medicine Inc.: Employment, Equity Ownership. Teixeira:Editas Medicine Inc.: Employment, Equity Ownership. Wilson:Editas Medicine Inc.: Employment, Equity Ownership. Albright:Editas Medicine Inc.: Employment, Equity Ownership. Jiang:Editas Medicine Inc.: Employment, Equity Ownership.

Background: Induction of fetal hemoglobin (HbF) is an attractive approach to ameliorate disease symptoms in beta hemoglobinopathies. Gene editing by zinc finger or CRISPR‐Cas nucleases to disrupt BCL11A expression, a known mediator of fetal globin silencing, via targeting BCL11A erythroid‐enhancer, is one approach to reactivate fetal globin expression. Alternatively, the genetics of hereditary persistence of fetal hemoglobin (HPFH) suggest that disruption of certain cis‐regulatory elements in the beta‐globin locus may also be a viable strategy. A previous lenti‐CRISPR mediated saturated mutagenesis screen identified multiple HbF‐inducing genomic domains concentrated at the gamma‐globin ( HBG1/2 ) promoters. Aims: To compare editing of HBG1/2 promoters versus BCL11A erythroid‐enhancer in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR‐Cas technologies as potential treatments for beta hemoglobinopathies. Methods: Ribonucleoprotein (RNP) complexes were designed to target either BCL11A erythroid‐enhancer GATAA motif or several HbF‐inducing genomic domains in the HBG1/2 promoters. These RNPs were transfected into mobilized peripheral blood (mPB) CD34+ HSPCs from healthy volunteers or peripheral blood mononuclear cells (PBMCs) from patients with sickle cell disease. RNPs demonstrated to have high HbF‐inducing potentials were used to edit HSPCs which were then infused into immunocompromised NOD.Cg‐Kit W−41J Tyr + Prkdc scid Il2rg tm1Wjl /ThomJ (NBSGW) mice to assess editing in SCID‐repopulating cells (SRC) and their multilineage reconstitution potential. Results: Following transfection of RNPs targeting HBG1/2 promoters into CD34+ HSPCs or sickle PBMCs, &gt;70% editing was achieved. Erythroid progeny derived from edited HSPCs or PBMCs had increased levels of HbF compared to those derived from control cells (up to 40% vs. 10% respectively). When infused into NBSGW mice, HSPCs edited with RNPs targeting either HBG1/2 promoters or BCL11A erythroid‐enhancer both reached &gt;70% human chimerism (hCD45+/hCD45+mCD45). Comparable monocytes, granulocytes, B lymphocytes, and HSPCs reconstitution were attained with both approaches. However, the erythroid lineage output in the BCL11A ‐edited group was approximately 80% less than that in the control group. In addition, a 20% reduction in editing levels, compounded by a 2.5‐fold higher nonproductive indel frequency, was observed in the erythroid compartment compared to unfractionated bone marrow (BM) or other human lineages from the same animal in the BCL11A ‐edited group. In contrast, HBG1/2 promoter‐edited cells had similar editing levels and indel patterns across all lineages with no significant lineage skewing. Importantly, robust long‐term induction of HbF was observed in human erythroid cells either sorted from chimeric BM or obtained from ex vivo culture of BM from HBG1/2 promoter‐edited group compared to the control group up to 16‐weeks post‐transplant. Summary/Conclusion: Our data suggest that sickle PBMCs, bulk CD34+ cells, and long‐term hematopoietic stem cells can be successfully edited using RNPs targeting BCL11A erythroid‐enhancer or HBG1/2 promoters. However, HBG1/2 promoter‐edited cells exhibited a more favorable preclinical profile than BCL11A ‐edited cells, as BCL11A ‐edited HSPCs displayed erythroid‐specific defects in the NBSGW mouse model. Long term induction of HbF by HBG1/2 promoter‐edited cells without lineage skewing suggests that this editing strategy has favorable clinical potential for the treatment of beta hemoglobinopathies.

Abstract Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (&amp;gt;70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) mediate their actions via a common G-protein-coupled receptor. High levels of PTH/PTHrP receptor expression have been detected in many tissues including bone and kidney. This study has demonstrated specific PTH/PTHrP receptor expression from the U3 promoter in the osteoblastic osteosarcoma ROS 17/2.8 cell line, which expresses the endogenous PTH/PTHrP receptor, compared to rat 2 fibroblasts which do not express the endogenous PTH/PTHrP receptor gene. Transient transfection studies revealed cell-specific expression of a construct containing 4391 bp of DNA upstream of exon U3 of the PTH/PTHrP receptor gene fused to a luciferase reporter gene. Deletion mapping of the 5′ region of U3 revealed that a construct containing 206 bp upstream of U3 confers cell-specific expression. These data suggest that cell-specific expression in ROS 17/2.8 involves cell-specific elements within the PTH/PTHrP receptor promoter.

We set out to determine the feasibility of using microarray-based resequencing for cancer gene mutation screening by designing GeneChip CustomSeq Resequencing arrays (Affymetrix, Santa Clara, CA, USA) for interrogation of ARAF, BRAF, CDK4, CDK6, CDKN2A, KLF6, HRAS, KRAS, MET, NRAS, PTEN, RAF1, RB1, RET and TP53 (164 exons in total). Arrays also included four intronic bases on either side of the exons to cover splice sites, thus the arrays covered a total of 23,966 bases. Overall performance was very good, with accuracy >99.99% and coverage ~97.5%. Twenty NSCLC samples were analyzed using the arrays, and several well-characterized somatic mutations and germline variants were found. The most significant novel finding was the detection of a transforming MET mutation (T1010I) in a NSCLC patient.

Microbial community (metagenomics) structure and function are known to play a significant role in human health, development and disease. Shotgun sequencing of metagenomes provides a catalog of genes in the microbial community. However, to understand function, it is necessary to interrogate the community transcriptome. Bacterial RNA content is >95% rRNA, and unlike eukaryotic mRNA, bacterial mRNA is not polyadenylated for easy isolation using oligo-dT selection. Therefore, it is essential to develop rRNA depletion protocols to make RNAseq cost effective. We are comparing different rRNA depletion methods: a hybrid subtraction-based approach using the MICROBExpress Kit (Ambion) and a 5'-phosphate-dependent exonuclease-based approach using the mRNA-ONLY™ Prokaryotic mRNA Isolation Kit (Epicentre). These depletion methods are being tested on well characterized genomes with a range of GC composition: P. marinus (30%), E. coli (51%), and R. sphaeroides (69%). MICROBExpress rRNA capture oligos were designed to work on intact E.coli 5S, 16S and 23S rRNA, however, all microbes do not have intact 16S and 23S: in R. sphaeroides, the 23S molecule is processed into separate 1.4kb and 1.6kb molecules, and in P.marinus, some of the 23S appears as smaller 0.7kb and 2.3kb molecules. Hence, different removal efficiencies have been observed. Visual inspection of the rRNA molecules on the Agilent Bioanalyzer shows almost complete depletion of 16S and 23S peaks for E.coli, depletion of the 16S peak for both R. sphaeroides and P. marinus but not the smaller 23S processed molecules in R. sphaeroides and P. marinus. By comparison, mRNA-ONLY removes roughly 2/3 of the rRNA transcripts in both E.coli and R. sphaeroides. To determine efficiency of mRNA enrichment using these methods, the mRNA was transcribed to cDNA and sequenced by Illumina. rRNA reads decreased by 25% in E.coli and 4.5% in R. sphaeroides with MICROBExpress and ~17% in both with mRNA-ONLY. As a result, a larger fraction of sequenced bases align to the annotated transcriptome with both methods: ~30-40% with MICROBExpress) and ~13-28% with mRNA-ONLY. We are evaluating additional depletion methods that will increase the amount of non-rRNA reads. These will also be applied to P. marinus. Determining enrichment efficiency by sequencing is very costly, thus we are developing qPCR assays for high, mid, and low expressed genes and the 16S and 23S rRNA genes for these organisms, and we will assess changes in rRNA/mRNA ratios with different depletion rRNA methods. The optimal method will be applied to microbe community transcriptomes.

Abstract The use of genetically engineered T cells expressing chimeric antigen receptors (CAR) against CD19 or BCMA to treat hematologic malignancies has demonstrated promising results in clinical trials. However, it is possible that the efficacy of CAR T cells could be limited in some clinical indications due to suppressive factors in the tumor microenvironment. Immunosuppressive cytokines (e.g., IL-6, IL-10 and TGFβ) provide a favorable environment for tumor growth and inhibit the tumoricidal activity of the endogenous T cells. In particular, transforming growth factor β (TGFβ) has been shown to suppress T-cell proliferation, inhibit the maturation of T helper cells and reduce their effector functions. Elevated levels of TGFβ have been detected in many human cancers, including multiple myeloma (MM). Here, we sought to modulate the activity of CAR T cell-based immunotherapy for MM by inhibiting TGFβ signaling in BCMA-specific CAR T cells (Budha et al., Cancer Cell, 2012). We evaluated two strategies to modulate the TGFβ pathway: (1) knocking out TGFβ Receptor II (TGFβRII) using CRISPR gene editing and (2) overexpressing a dominant negative TGFβRII in BCMA targeted CAR T cells. TGFβRII modulation inhibited TGFβ-dependent Smad2/3 phosphorylation. TGFβRII-modulated BCMA CAR T cells were insensitive to TGFβ-mediated inhibition of proliferation, cell cytotoxicity and cytokine production, both IFNg and granzyme B, in the context of primary and/or repeat antigen stimulation with BCMA+ve MM cell lines in vitro . We further evaluated the gene expression signatures of BCMA CAR T cells isolated from tumor and spleen in a NSG mouse xenograft model subcutaneously inoculated with myeloma cell line (RPMI8226). TGFβ pathway gene expression changes indicating TGFβ exposure were observed in BCMA CAR T cells isolated from tumor, but not spleen, suggesting that BCMA CAR T cells are encountering a TGFβ-enriched TME. However, both TGFβ pathway modulating strategies prevented the development of the TGFβ-induced gene expression phenotype. Together, these data suggest that inhibition of TGFβRII signaling may potentiate CAR T activity by directly inhibiting immunosuppressive effects of TGFβ in the tumor microenvironment. By specifically targeting TGFβ signaling in CAR T cells, it may be possible to avoid side effects associated with systemic administration of TGFβ antagonists. Gene editing to remove TGFβRII expression or overexpressing dominant negative TGFβ RII could modulate the activity of BCMA CAR T therapy in MM. Disclosures Vong: Juno Therapeutics: Employment, Equity Ownership. Nye: Juno Therapeutics: Employment, Equity Ownership. Hause: Juno Therapeutics: Employment, Equity Ownership. Clouser: Juno Therapeutics: Employment, Equity Ownership. Jones: Juno Therapeutics: Employment, Equity Ownership. Burleigh: Juno Therapeutics: Employment, Equity Ownership. Borges: Editas Medicine: Employment, Equity Ownership. Chin: Editas Medicine: Employment, Equity Ownership. Marco: Editas Medicine: Employment, Equity Ownership. Barrera: Editas Medicine: Employment, Equity Ownership. Da Silva: Editas Medicine: Employment, Equity Ownership. Harbinski: Editas Medicine: Employment, Equity Ownership. Giannoukos: Editas Medicine: Employment, Equity Ownership. Dhanapal: Editas Medicine: Employment, Equity Ownership. Jiang: Juno Therapeutics: Employment, Equity Ownership. Salmon: Juno Therapeutics: Employment, Equity Ownership. Wilson: Editas Medicine: Employment, Equity Ownership. Myer: Editas Medicine: Employment, Equity Ownership. Welstead: Editas Medicine: Employment, Equity Ownership. Bond: Juno Therapeutics: Employment, Equity Ownership. Sather: Juno Therapeutics: Employment, Equity Ownership.

Figure S8. Comparison of Indel rates between UDiTaS and other methods. T-Cells edited with the TRAC + B2M guides were analyzed for indel editing at the TRAC locus using PCR-amplification followed by Sanger Sequencing or NGS, in addition to UDiTaS with two different anchor primers. Indel rates were very similar between the methods. (PPTX 81Â kb)