The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

The new frontier of genome engineering with CRISPR-Cas9

Stem cells are defined as cells able to both extensively self-renew and differentiate into progenitors. Research data show that more resistant stem cells than common cancer cells exist in cancer patients.To identify unrecognized differences between cancer stem cells and cancer cells might be able to develope effective classification,diagnose and treat ment for cancer.

Stem Cells,Cancer and Cancer Stem Cells

Tumour formation involves the co-evolution of neoplastic cells together with extracellular matrix, tumour vasculature and immune cells Successful outgrowth of tumours and eventual metastasis is not determined solely by genetic alterations in tumour cells, but also by the fitness advantage such mutations confer in a given environment As fitness is context dependent, evaluating tumours as complete organs, and not simply as masses of transformed epithelial cells, becomes paramount The dynamic tumour topography varies drastically even throughout the same lesion Heterologous cell types within tumours can actively influence therapeutic response and shape resistance

Influence of tumour micro-environment heterogeneity on therapeutic response

Clonal Heterogeneity and Tumor Evolution: Past, Present, and the Future.

Iterative model-building, structure refinement, and density modification with the PHENIX AutoBuild Wizard Thomas C. Terwilliger a* , Ralf W. Grosse-Kunstleve b , Pavel V. Afonine b , Nigel W. Moriarty b , Peter Zwart b , Li-Wei Hung a , Randy J. Read c , Paul D. Adams b* a b Los Alamos National Laboratory, Mailstop M888, Los Alamos, NM 87545, USA Lawrence Berkeley National Laboratory, One Cyclotron Road, Bldg 64R0121, Berkeley, CA 94720, USA. c Department of Haematology, University of Cambridge, Cambridge CB2 0XY, UK. * Email: terwill@lanl.gov or PDAdams@lbl.gov Running title: The PHENIX AutoBuild Wizard Abstract The PHENIX AutoBuild Wizard is a highly automated tool for iterative model- building, structure refinement and density modification using RESOLVE or TEXTAL model- building, RESOLVE statistical density modification, and phenix.refine structure refinement. Recent advances in the AutoBuild Wizard and phenix.refine include automated detection and application of NCS from models as they are built, extensive model completion algorithms, and automated solvent molecule picking. Model completion algorithms in the AutoBuild Wizard include loop-building, crossovers between chains in different models of a structure, and side-chain optimization. The AutoBuild Wizard has been applied to a set of 48 structures at resolutions ranging from 1.1 A to 3.2 A, resulting in a mean R-factor of 0.24 and a mean free R factor of 0.29. The R-factor of the final model is dependent on the quality of the starting electron density, and relatively independent of resolution. Keywords: Model building; model completion; macromolecular models; Protein Data Bank; structure refinement; PHENIX Introduction Iterative model-building and refinement is a powerful approach to obtaining a complete and accurate macromolecular model. The approach consists of cycles of building an atomic model based on an electron density map for a macromolecular structure, refining the structure, using the refined structure as a basis for improving the map, and building a new model. This type of approach has been carried out in a semi-automated fashion for many years, with manual model-building iterating with automated refinement (Jensen, 1997). More recently, with the development first of ARP/wARP (Perrakis et al., 1999), and later other procedures including RESOLVE iterative model-building and refinement (Terwilliger,

Iterative model-building, structure refinement, and density modification with the PHENIX AutoBuild Wizard

AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

/pdf/rna-directed-gene-editing-specifically-eradicates-latent-and-1l8k0070j9.pdf

RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection

In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models.

Ras Induces Mediator Complex Exchange on C/EBPβ

Gastric cancer is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. To date, there is a lack of efficient therapeutic protocols for gastric cancer. Recent studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and resistance to anticancer therapies. Thus, therapies that target gastric CSCs are attractive. However, CSCs in human gastric adenocarcinoma (GAC) have not been described. Here, we identify CSCs in tumor tissues and peripheral blood from GAC patients. CSCs of human GAC (GCSCs) that are isolated from tumor tissues and peripheral blood of patients carried CD44 and CD54 surface markers, generated tumors that highly resemble the original human tumors when injected into immunodeficient mice, differentiated into gastric epithelial cells in vitro, and self-renewed in vivo and in vitro. Our findings suggest that effective therapeutic protocols must target GCSCs. The capture of GCSCs from the circulation of GAC patients also shows great potential for identification of a critical cell population potentially responsible for tumor metastasis, and provides an effective protocol for early diagnosis and longitudinal monitoring of gastric cancer.

/pdf/identification-and-expansion-of-cancer-stem-cells-in-tumor-1nuj01vky5.pdf

Identification and expansion of cancer stem cells in tumor tissues and peripheral blood derived from gastric adenocarcinoma patients

Serum tumor biomarker carbohydrate antigen 724 (CA724) is noticeable for gastric cancer. Correlation between CA724 and gastric cancer was investigated based on Chinese population. Chinese Biomedical Database, Chinese Journal Full-text Database and PubMed were searched. Gastric cancer patients were proven by biopsy, and control included health volunteers or benign gastric diseases. Participants received at least one test of CA724, CA125, CA153, CA199, CA242 or CEA. Meta-analysis, summary ROC (SROC) and post hoc analysis were performed by RevMan 5.0 and SPSS 11.5. Totally, 33 eligible studies were analyzed. Meta-analysis showed CA724 had the highest odds ratio 32.86 compared to control, orderly followed by CA242, CA199, CEA, CA125 and CA153. Accumulated accuracy rate of CA724 was 77 %, superior to others. In SROC analysis, specificity of all studies was above 0.70, but sensitivity of few studies was above 0.70; CA724 was selected as the preferable single test, followed by CA242, CA199, CEA, CA125 and CA153. If threshold of both specificity and sensitivity up to 0.70, CA153 was unacceptable; if up to 0.80, only CA724 and CA242 were considerable. In CA724-combined patterns, CA724+CEA+CA199 combination performed best by increasing sensitivity to 0.74 without impairing specificity, while CA724 + CA199 pattern was not a proper combination. CA724 was the most correlative serum tumor biomarker for gastric cancer in Chinese population. Sensitivity of serum CA724 is limited, but CA724+CEA+CA199 combination is considerable to improve sensitivity without impairing specificity.

Xianming Mo

Papers

RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection

In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models.

Ras Induces Mediator Complex Exchange on C/EBPβ

Identification and expansion of cancer stem cells in tumor tissues and peripheral blood derived from gastric adenocarcinoma patients

Correlation between serum CA724 and gastric cancer: multiple analyses based on Chinese population