CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system.

Existing methods to enrich target regions of genomic DNA based on PCR, hybridization capture, or molecular inversion probes have various drawbacks, including long experiment times and low throughput and/or enrichment quality. We developed CRISPR-Cap, a simple and scalable CRISPR-based method to enrich target regions of dsDNA, requiring only two short experimental procedures that can be completed within two hours. We used CRISPR-Cap to enrich 10 target genes 355.7-fold on average from Escherichia coli genomic DNA with a maximum on-target ratio of 81% and high enrichment uniformity. We also used CRISPR-Cap to measure gene copy numbers and detect rare alleles with frequencies as low as 1%. Finally, we enriched coding sequence regions of 20 genes from the human genome. We envision that CRISPR-Cap can be used as an alternative to other widely used target-enrichment methods, which will broaden the scope of CRISPR applications to the field of target enrichment field.

Highly selective retrieval of accurate DNA utilizing a pool of in situ-replicated DNA from multiple next-generation sequencing platforms.

Scalable and cost-effective production of error-free DNA is critical to meet the increased demand for such DNA in the field of biological science. Methods based on 'Dial-out PCR' have enabled the high-throughput error-free DNA synthesis from a microarray-synthesized DNA pool by labeling with retrieval PCR tags, and retrieving error-free DNA of which the sequence is identified via next generation sequencing (NGS). However, most of the retrieved products contain byproducts due to background amplification of redundantly labeled DNAs. Here, we present a highly selective retrieval method of desired DNA from a pool of millions of DNA clones from NGS platforms. Our strategy is based on replicating entire sequence-verified DNA molecules from NGS plates to obtain population-controlled DNA pool. Using the NGS-replica pool, we could perform improved and selective retrieval of desired DNA from the replicated DNA pool compared to other dial-out PCR based methods. To evaluate the method, we tested this strategy by using 454, Illumina, and Ion Torrent platforms for producing NGS-replica pool. As a result, we observed a highly selective retrieval yield of over 95%. We anticipate that applications based on this method will enable the preparation of high-fidelity sequenced DNA from heterogeneous collections of DNA molecules.

Tumor evolution and intratumor heterogeneity of an epithelial ovarian cancer investigated using next-generation sequencing.

BACKGROUND: The extent to which metastatic tumors further evolve by accumulating additional mutations is unclear and has yet to be addressed extensively using next-generation sequencing of high-grade serous ovarian cancer. METHODS: Eleven spatially separated tumor samples from the primary tumor and associated metastatic sites and two normal samples were obtained from a Stage IIIC ovarian cancer patient during cytoreductive surgery prior to chemotherapy. Whole exome sequencing and copy number analysis were performed. Omental exomes were sequenced with a high depth of coverage to thoroughly explore the variants in metastatic lesions. Somatic mutations were further validated by ultra-deep targeted sequencing to sort out false positives and false negatives. Based on the somatic mutations and copy number variation profiles, a phylogenetic tree was generated to explore the evolutionary relationship among tumor samples. RESULTS: Only 6% of the somatic mutations were present in every sample of a given case with TP53 as the only known mutant gene consistently present in all samples. Two non-spatial clusters of primary tumors (cluster P1 and P2), and a cluster of metastatic regions (cluster M) were identified. The patterns of mutations indicate that cluster P1 and P2 diverged in the early phase of tumorigenesis, and that metastatic cluster M originated from the common ancestral clone of cluster P1 with few somatic mutations and copy number variations. CONCLUSIONS: Although a high level of intratumor heterogeneity was evident in high-grade serous ovarian cancer, our results suggest that transcoelomic metastasis arises with little accumulation of somatic mutations and copy number alterations in this patient.

Globally shared TCR repertoires within the tumor-infiltrating lymphocytes of patients with metastatic gynecologic cancer.

Gynecologic cancer, including ovarian cancer and endometrial cancer, is characterized by morphological and molecular heterogeneity. Germline and somatic testing are available for patients to screen for pathogenic variants in genes such as BRCA1/2. Tissue expression levels of immunogenomic markers such as PD-L1 are also being used in clinical research. The basic therapeutic approach to gynecologic cancer combines surgery with chemotherapy. Immunotherapy, while not yet a mainstream treatment for gynecologic cancers, is advancing, with Dostarlimab recently receiving approval as a treatment for endometrial cancer. The goal remains to harness stimulated immune cells in the bloodstream to eradicate multiple metastases, a feat currently deemed challenging in a typical clinical setting. For the discovery of novel immunotherapy-based tumor targets, tumor-infiltrating lymphocytes (TILs) give a key insight on tumor-related immune activities by providing T cell receptor (TCR) sequences. Understanding the TCR repertoires of TILs in metastatic tissues and the circulation is important from an immunotherapy standpoint, as a subset of T cells in the blood have the potential to help kill tumor cells. To explore the relationship between distant tissue biopsy regions and blood circulation, we investigated the TCR beta chain (TCRß) in bulk tumor and matched blood samples from 39 patients with gynecologic cancer. We found that the TCR clones of TILs at different tumor sites were globally shared within patients and had high overlap with the TCR clones in peripheral blood.

Engineered Attenuated Salmonella typhimurium Expressing Neoantigen Has Anticancer Effects.

Neoantigen vaccines are an immunotherapy strategy for treating cancer. The vaccine degrades quickly, so the strategy must include protection and precise targeting for immune cell stimulation. In this study, we engineered attenuated Salmonella typhimurium, which is highly infiltrative to tumors, to act as a carrier for Neoantigen peptide vaccine. Our system used a constitutive promoter vector, so that a single injection of Salmonella expressing Neoantigen could be used without requiring additional induction injections. In vivo experiments on bacteria-treated mice showed that Neoantigen expressed by the engineered carrier infiltrated tumors and resulted in suppressed tumor growth, higher survival rates and longer survival times, a relative increase of CD4 and CD8 T cells, and cytokine release. These results indicate that engineered Salmonella can be used as a carrier for Neoantigen immunotherapy.

Novel somatic variants involved in biochemical activity of pure growth hormone-secreting pituitary adenoma without GNAS variant.

We aimed to identify somatic genetic alterations in pure growth hormone (GH)-secreting pituitary adenomas without GNAS variants. Patients with GH-secreting pituitary adenoma who underwent transsphenoidal adenomectomy at Severance Hospital, Yonsei University College of Medicine were recruited. Somatic genetic alterations were profiled by whole-exome sequencing (WES) and targeted resequencing. WES was performed using DNA from nine GH-secreting pituitary tumors and corresponding blood samples. Absence of GNAS variant was confirmed by Sanger sequencing. For targeted resequencing of 140 fixed tissues, 48 WES-derived candidate genes and 7 GH-secreting pituitary adenoma-associated genes were included. Forty-eight genes with 59 somatic variants were identified by WES. In targeted resequencing, variants in 26 recurrent genes, including MAST4, PRIM2, TNN, STARD9, DNAH11, DOCK4, GPR98, BCHE, DARS, CUBN, NGDN, PLXND1, UNC5B, and COL22A1, were identified, but variants in previously reported genes were not detected. BCHE, DARS, NGDN, and UNC5B variants were associated with increased GH-secreting pituitary tumor biochemical activity, which was confirmed in vitro. Although recurrent point variants were rare, several somatic variants were identified in sporadic pure GH-secreting pituitary adenomas. Several somatic variants may affect pathways involved in the tumorigenesis and biochemical activities of GH-secreting pituitary adenomas.

Single-cell analysis of a mutant library generated using CRISPR-guided deaminase in human melanoma cells.

CRISPR-based screening methods using single-cell RNA sequencing (scRNA-seq) technology enable comprehensive profiling of gene perturbations from knock-out mutations. However, evaluating substitution mutations using scRNA-seq is currently limited. We combined CRISPR RNA-guided deaminase and scRNA-seq technology to develop a platform for introducing mutations in multiple genes and assessing the mutation-associated signatures. Using this platform, we generated a library consisting of 420 sgRNAs, performed sgRNA tracking analysis, and assessed the effect size of the response to vemurafenib in the human melanoma cell line, which has been well-studied via knockout-based drop-out screens. However, a substitution mutation library screen has not been applied and transcriptional information for mechanisms of action was not assessed. Our platform permits discrimination of several candidate mutations that function differently from other mutations by integrating sgRNA candidates and gene expression readout. We anticipate that our platform will enable high-throughput analyses of the mechanisms related to a variety of biological events.

Multiplex Generation, Tracking, and Functional Screening of Substitution Mutants Using a CRISPR/Retron System.

We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/retron system for multiplexed generation of substitution mutations by coutilization of a retron system that continuously expresses donor DNA and a CRISPR/Cas9 cassette that induces cleavage at target genomic loci. Our system efficiently introduces substitution mutation in the Escherichia coli genome in a high-throughput manner. These substitution mutations can be tracked by analysis of retron plasmid sequences without laborious amplification of individual edited loci. We demonstrated that our CRISPR/retron system can introduce thousands of mutations in a single experiment and be used for screening phenotypes related to chemical responses or fitness changes. We expect that our system could facilitate genome-scale substitution screenings.

Straightforward Delivery of Linearized Double-Stranded DNA Encoding sgRNA and Donor DNA for the Generation of Single Nucleotide Variants Based on the CRISPR/Cas9 System.

CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination. Typically, single-stranded oligodeoxynucleotide, serving as the donor template, and a plasmid encoding guide RNA are delivered as two separate components. However, in the multiplexed generation of single nucleotide variants, this two-component delivery system is limited by difficulty of delivering a matched pair of sgRNA and donor DNA to the target cell. Here, we describe a novel codelivery system called "sgR-DNA" that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA. Our sgR-DNA-based method is simple to implement because it does not require cloning steps. We also report the potential of our delivery system to generate multiplex genomic substitutions in Escherichia coli and human cells.