CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

Study of comparative proteome between normal and inverted karyotypes of human mesenchymal stem cells

Multipotent mesenchymal stem cells have been expanded in vitro for cellular therapy in numerous clinical settings without standardized culture conditions or quality-control schemes. The in vitro expansion is necessary to obtain sufficient cells for clinical applications. However, the expansion may induce genetic and functional abnormalities which may affect the safety and functionality of MSC, especially the chromosomal stability. This study aimed to investigate the protein profile of umbilical cord-derived MSC with normal and inverted karyotypes after expansion in the laboratory. Mass spectrometry analysis was performed and the Bradford method, Scaffold software, String and Cytoscape databases were employed to measure and characterize the protein content of umbilical cord-derived MSC. Networks of protein interactions, hub and bottleneck proteins were identified by proteomics and systems biology approaches. We found that proteins related to cellular stress were super expressed in inverted karyotype cells. Moreover, a high expression of Serpine 1, RHOA, and CTSB was found in these cells, which are proteins related to cancer. The albumin and ubiquitin proteins have been associated with a positive prognosis in cancer and cellular stress, and were up- and down-regulated in normal karyotype cells, respectively. The results suggests that the paracentric inversion inv(3)(p25p13) induced some type of cellular stress and genetic instability in human mesenchymal stem cells. These analyses showed the importance of carrying out studies related to the genetic instability of human mesenchymal stem cells using the protein expression profile as a parameter.

Role of gastrointestinal microbiota in tumorigenesis / 上海交通大学学报（医学版）

Gastrointestinal microbiota has an important impact on physiological functions of human body. In recent years,the importance of gastrointestinal microbiota in tumorigenesis has emerged. Aberrant location and proportion of microbiota can cause not only carcinogenesis of local epithelia,but also tumorigenesis in submucous tissues or even distant organs. Certain microbes can produce genotoxin or generate reactive oxygen species to cause DNA damage and affect DNA damage repair,which leads to genetic instability and induces cell transformation. Moreover,gastrointestinal microbiota is able to affect the function of host immune system and form an immunosuppressive microenvironment,while some other pathogenic bacteria can cause chronic inflammation and may be involved in tumor immune escape. Based on the above,gastrointestinal microbiota is oncogenic and closely linked to tumorigenesis.

Caracterización molecular del gen supresor de tumores TP53 en el cáncer colorrectal / Molecular characterization of TP53 tumor suppressor gene in colorectal cancer

Introducción: El cáncer colorrectal (CCR) es una de las neoplasias más comunes en el mundo; especialmente,en los países desarrollados. En Colombia, la incidencia del CCR ocupa el cuarto lugar en hombres ymujeres; el CCR tiene una gran heterogeneidad genética. Objetivo: Determinar la presencia de mutacionesen los exones 5-8 del gen TP53 en tumores colorrectales, mediante el secuenciamiento directo. Métodos:Muestras con diagnóstico histopatológico de CCR esporádico se dividieron en dos grupos. El Grupo I fue de 30 muestras de tumores a partir de biopsias frescas, y el Grupo II, de 46 muestras de tejidos tumorales embebidos en bloques de parafi na. El análisis de mutaciones se realizó en los exones 5-8 del gen TP53,empleando las técnicas de PCR y de secuenciamiento directo. Resultados: Se encontró una baja frecuenciade mutaciones en el gen TP53, del 4,4%; las mutaciones detectadas fueron sin sentido; además, fueronidentifi cados dos polimorfi smos que segregan juntos. Todas las mutaciones y los polimorfi smos se detectaron en las muestras del grupo I. La mayoría de las muestras analizadas se hallaban en un estado avanzado del cáncer. Conclusiones: La baja frecuencia obtenida de mutaciones en TP53 permite sugerir la existencia de alteraciones en otras vías genéticas, relacionadas con la carcinogénesis colorrectal, como las vías de MSI y de CIN, así como la epigenética; dichas alteraciones no podrían excluirse en las muestras evaluadas. Los estudios moleculares en muestras de tejidos embebidos en parafi na presentan difi cultades para los análisis genéticos. La caracterización molecular del CCR es importante para conocer el espectro de mutaciones y de variantes moleculares presentes en la población observada.

Introduction: Colorectal cancer (CRC) is one of the most common malignancies in the world, especially indeveloped countries. In Colombia, the incidence of CRC ranks fourth in men and women. CRC has greatgenetic heterogeneity. Purpose: The purpose of this study was to determine the presence of mutations inexons 5 to 8 of the TP53 gene in colorectal tumors by direct sequencing. Patients and Methods: Samples with histopathological diagnoses of sporadic CRC were divided into two groups. Group I included 30 tumor samples from fresh biopsies and Group II included 46 tumor tissue samples embedded in paraffi n blocks. Mutational analysis was performed for exons 5 through 8 of the TP53 gene using PCR and direct sequencing.Results: The frequency of TP53 mutations was only 4.4%, and mutations that were detected were nonsense mutations. In addition, two polymorphisms that segregate together were identifi ed. All mutations and polymorphismswere detected in samples from Group I. Most of the samples were in advanced stages of cancer.Conclusions: The low frequency of mutations in TP53 suggests the existence of alterations on other related genetic pathways in colorectal carcinogenesis. These could include MSI pathways, CIN and epigenetics. Such alterations could not be excluded in the samples tested. Molecular studies of tissue samples embedded inparaffi n are diffi cult to analyze genetically. Molecular characterization of CRC is important for determining the spectrum of mutations and molecular variants present in our population.

Understanding of molecular pathogenesis and genetic markers in colorectal cancer / 대한내과학회지

Colorectal cancers results from the progressive accumulation of genetic and epigenetic alterations that lead to cellular transformation and tumor progression. Genomic instability, including chromosomal translocations and microsatellite instability, plays a role in acquisition of enough mutations for malignant transformation. In addition, epigenetic silencing is an important mechanism in the evolution of a subgroup of colorectal cancers. These genetic and epigenetic changes causes activation of oncogene pathway (APC, KRAS) and inactivation of tumor-suppressor pathway (p53, TGF-beta). Recent advance in colorectal carcinogenensis leads to development of molecular markers for early detection and predictive and prognostic markers.

Genetic Instability in Gastric Epithelial Neoplasias Categorized by the Revised Vienna Classification

BACKGROUND/AIMS: The aim of this study was to determine the structural chromosomal aberrations, such as loss of heterozygosity (LOH) and microsatellite instability (MSI), at multiple tumor suppressor gene loci in gastric epithelial neoplasia categorized by the revised Vienna classification. METHODS: All tissue samples were excised by endoscopic mucosal resection. Sixty category 3 (low-grade adenoma) tissue samples and 51 category 4 samples (high-grade adenoma and intramucosal carcinoma with adenoma) were examined at the 7 sets of microsatellite loci linked to the tumor suppressor gene locus. RESULTS: For category 3 and 4 tissue samples, there were no differences in the frequencies of LOH-positive chromosomes or the extent of chromosomal loss. The Helicobacter-pylori (H. pylori)-positive rate was significantly higher in MSI-positive category 4 samples than in category 3 samples (p=0.04). The frequency of MSI positivity was significantly higher in category 4 samples than in category 3 samples (p=0.003). CONCLUSIONS: H. pylori infection is associated with genetic instability of the premalignant lesion. MSI occurs in the early stages of gastric carcinogenesis and its occurrence increases during malignant transformation. Detection of MSI in premalignant gastric lesions may be a surveillant of risk of malignant transformation.

Genetic instability of P57~(kip2) misexpression in human hepatocellular carcinoma / 基础医学与临床

Objective To study the expressions of P57~(kip2) mRNA and genetic instability of P57~(kip2) in human hepatocellular carcinoma (HCC).Methods In situ hybridization(ISH) was used to detect the expression of P57~(kip2) mRNA in HCC.MSI and LOH were detected by PCR-polyacrylamide gel electmphoresis-silver staining method.Results There was no expression of P57~(kip2) mRNA found in normal liver tissue.The expression rate of P57~(kip2) mRNA in both pericancerous cirrhosis and hepatocellular carcinoma was 26.7%(8/30).LOH was not identified in 30 cases on three microsatellite loeies;there were 2 microsatellite loeies showing MSI,the total rate of MSI was 16.7%.There was correlation found between the MSI of D11S1760 locies and the expression of P57~(kip2) mRNA(P＜0.05).Conclusion The disorder expression of P57~(kip2) mRNA indicated that P57~(kip2) might be involved in hepatocarcinogenesis and prognosis.MSI may be one of the reasons that explains the disorder expression of P57~(kip2) mRNA in hepatocarcinogenesis and prognosis.

Genetic Instability and Mutations of Mismatch Repair (MMR) and p53 Gene in Colorectal Cancers with Multiple Polyps and Sporadic Colorectal Cancers

PURPOSE: General conceptions of carcinogenic mechanisms by recent reports are ras-p53 gene pathway in sporadic colorectal cancers (SCC), MMR gene pathway in hereditary nonpolyposis colorectal cancer (HNPCC) and APC gene in familial adenomatous polyposis (FAP). But in the colorectal cancer with multiple polyps (CCMP), the carcinogenic pathway is not still defined exactly. In order to find out the which carcinogenic pathway control the CCMP and SCC, genetic instability were studied in CCMP and SCC. METHODS: In this study, genetic instability on D2S123, D3S1029, D5S107, D6S87 and AP delta3 foci and gene mutations of hMLH1 (exon 2, 16, 19), hMSH2 (exon 11, 12, 13, 14) gene of MMR gene, p53 gene (exon 5, 6, 7, 8, 9) were studied on the 60 DNA samples of CCMP (30 cases) and SCC (30 cases). RESULTS: 1. Observed positive genetic instability was higher in CCMP (30%) than SCC (20%), and was higher in right colon cancers (33%) than left colon cancers (23%) or rectal cancers (17%), but not significant statisitically. And observed positive genetic instability was lower in moderate differentiated cancers (16%) than well (67%) or poorly (60%) differentiated cancers (P=0.005). 2. Any mutations of hMLH1 and hMSH2 gene of MMR gene were not observed in both of CCMP and SCC, but 3 cases (2 CCMPs and 1 SCC) point mutations of intron of hMSH2 gene, which were higher in positive genetic instability than negative (P=0.002). 3. This 3 cases point mutations were C for T which were on 6th bases upstream from codon 669. 4. From the results of SSCP for nucleotide sequencing of p53 gene, the abnormal bands were observed in 30% (9 of 30) of CCMP and SCC. Also the abnormal bands were observed in both of positive or negative genetic instability without differences. CONCLUSIONS: With above results the authors suggested that the mechanism of genetic instability and mutations of p53 gene strongly affect the mechanism of carcinogenesis in SCC and CCMP. And there are relationship between genetic instability and point mutation at intron region of hMSH2 gene. However further evaluation and research is needed to establish relation between APC gene and other different kind of MMR gene.