A highly efficient transgene knock-in technology in clinically relevant cell types.

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

Spatially distinct physiology of Bacteroides fragilis within the proximal colon of gnotobiotic mice.

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.

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

UDiTaS™, a genome editing detection method for indels and genome rearrangements.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Simultaneous generation of many RNA-seq libraries in a single reaction.

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.

Functional optimization of gene clusters by combinatorial design and assembly.

Large microbial gene clusters encode useful functions, including energy utilization and natural product biosynthesis, but genetic manipulation of such systems is slow, difficult and complicated by complex regulation. We exploit the modularity of a refactored Klebsiella oxytoca nitrogen fixation (nif) gene cluster (16 genes, 103 parts) to build genetic permutations that could not be achieved by starting from the wild-type cluster. Constraint-based combinatorial design and DNA assembly are used to build libraries of radically different cluster architectures by varying part choice, gene order, gene orientation and operon occupancy. We construct 84 variants of the nifUSVWZM operon, 145 variants of the nifHDKY operon, 155 variants of the nifHDKYENJ operon and 122 variants of the complete 16-gene pathway. The performance and behavior of these variants are characterized by nitrogenase assay and strand-specific RNA sequencing (RNA-seq), and the results are incorporated into subsequent design cycles. We have produced a fully synthetic cluster that recovers 57% of wild-type activity. Our approach allows the performance of genetic parts to be quantified simultaneously in hundreds of genetic contexts. This parallelized design-build-test-learn cycle, which can access previously unattainable regions of genetic space, should provide a useful, fast tool for genetic optimization and hypothesis testing.

Relating the metatranscriptome and metagenome of the human gut.

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.

Non-invasive mapping of the gastrointestinal microbiota identifies children with inflammatory bowel disease.

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.

Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes.

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.

Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons.

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.

Analysis of exonic elastin variants in severe, early-onset chronic obstructive pulmonary disease.

The destruction of elastic fibers has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Emphysema has been described in autosomal dominant cutis laxa, which can be caused by mutations in the elastin gene. Previously, a rare functional mutation in the terminal exon of elastin was found in a case of severe, early-onset COPD. To test the hypothesis that other similar elastin mutations may predispose to COPD, we screened 90 probands from the Boston Early-Onset COPD Study and 90 smoking control subjects from the Normative Aging Study for mutations in elastin exons using high-resolution DNA melt analysis followed by resequencing. Rare nonsynonymous single-nucleotide polymorphisms (SNPs) seen only in cases were examined for segregation with airflow obstruction within pedigrees. Common nonsynonymous SNPs were tested for association with COPD in a family-based analysis of 949 subjects from the Boston Early-Onset COPD Study, and in a case-control analysis in 389 COPD cases from the National Emphysema Treatment Trial and 472 control subjects from the Normative Aging Study. Of 28 elastin variants found, 3 were nonsynonymous SNPs found only in cases. The previously described Gly773Asp mutation was found in another proband. The other two SNPs did not clearly segregate with COPD within families. Two common nonsynonymous SNPs did not demonstrate significant associations in either a family-based or case-control analysis. Exonic SNPs in the elastin gene do not appear to be common risk factors for severe COPD.

High-resolution melting curve analysis of genomic and whole-genome amplified DNA.

BACKGROUND: High-resolution melting curve analysis is an accurate method for mutation detection in genomic DNA. Few studies have compared the performance of high-resolution DNA melting curve analysis (HRM) in genomic and whole-genome amplified (WGA) DNA. METHODS: In 39 paired genomic and WGA samples, 23 amplicons from 9 genes were PCR amplified and analyzed by high-resolution melting curve analysis using the 96-well LightScanner (Idaho Technology). We used genotyping and bidirectional resequencing to verify melting curve results. RESULTS: Melting patterns were concordant between the genomic and WGA samples in 823 of 863 (95%) analyzed sample pairs. Of the discordant patterns, there was an overrepresentation of alternate melting curve patterns in the WGA samples, suggesting the presence of a mutation (false positives). Targeted resequencing in 135 genomic and 136 WGA samples revealed 43 single nucleotide polymorphisms (SNPs). All SNPs detected in genomic samples were also detected in WGA. Additional genotyping and sequencing allowed the classification of 628 genomic and 614 WGA amplicon samples. Heterozygous variants were identified by non-wild-type melting pattern in 98% of genomic and 97% of WGA samples (P = 0.11). Wild types were correctly classified in 99% of genomic and 91% of WGA samples (P < 0.001). CONCLUSIONS: In WGA DNA, high-resolution DNA melting curve analysis is a sensitive tool for SNP discovery through detection of heterozygote variants; however, it may misclassify a greater number of wild-type samples.