Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (pooled CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells and present a framework to power biological discovery with the resulting genotype-phenotype map. We use transcriptional phenotypes to predict the function of poorly-characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena - from RNA processing to differentiation. We leverage this ability to systematically identify the genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene function and cellular behavior.

Genomic Mapping in Detection of Vascular Disorders

Term vascular dysfunctional refers to a wide spectrum of vascular abnormalities including pathogenesis of tumors, their proliferation and leading to malfunctioned conditions. The treatment of most of the vascular anomalies including peripheral arterial disease and cardiac disease is multi factor procedure which include the embolic therapy, laser-based treatments and coagulation are all plating important role in managing the disease associated with vascular breakdown. The research proposal defines the treatment and diagnosis procedures involved in treatment of vascular abnormalities with a deep emphasis on techniques, efficiency and complications resulted from various other procedure and use of mapping and sequencing techniques based on genetics of variants of selected genes PIk3CA which will ultimately be more effective.

Development of Methods Derived from Iodine-Induced Specific Cleavage for Identification and Quantitation of DNA Phosphorothioate Modifications

DNA phosphorothioate (PT) modification is a novel modification that occurs on the DNA backbone, which refers to a non-bridging phosphate oxygen replaced by sulfur. This exclusive DNA modification widely distributes in bacteria but has not been found in eukaryotes to date. PT modification renders DNA nuclease tolerance and serves as a constitute element of bacterial restriction–modification (R–M) defensive system and more biological functions are awaiting exploration. Identification and quantification of the bacterial PT modifications are thus critical to better understanding their biological functions. This work describes three detailed methods derived from iodine-induced specific cleavage-an iodine-induced cleavage assay (ICA), a deep sequencing of iodine-induced cleavage at PT site (ICDS) and an iodine-induced cleavage PT sequencing (PT-IC-Seq)-for the investigation of PT modifications. Using these approaches, we have identified the presence of PT modifications and quantized the frequency of PT modifications in bacteria. These characterizations contributed to the high-resolution genomic mapping of PT modifications, in which the distribution of PT modification sites on the genome was marked accurately and the frequency of the specific modified sites was reliably obtained. Here, we provide time-saving and less labor-consuming methods for both of qualitative and quantitative analysis of genomic PT modifications. The application of these methodologies will offer great potential for better understanding the biology of the PT modifications and open the door to future further systematical study.

Intestinal Microbiota in Colorectal Cancer Surgery

The intestinal microbiota consists of numerous microbial species that collectively interact with the host, playing a crucial role in health and disease. Colorectal cancer is well-known to be related to dysbiotic alterations in intestinal microbiota. It is evident that the microbiota is significantly affected by colorectal surgery in combination with the various perioperative interventions, mainly mechanical bowel preparation and antibiotic prophylaxis. The altered postoperative composition of intestinal microbiota could lead to an enhanced virulence, proliferation of pathogens, and diminishment of beneficial microorganisms resulting in severe complications including anastomotic leakage and surgical site infections. Moreover, the intestinal microbiota could be utilized as a possible biomarker in predicting long-term outcomes after surgical CRC treatment. Understanding the underlying mechanisms of these interactions will further support the establishment of genomic mapping of intestinal microbiota in the management of patients undergoing CRC surgery.