Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5′-NNNVRYM-3′ as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering. Keywords: CRISPR-Cas system; Cas9; protospacer adjacent motif; RNA triplex; crystal structureUnited States. Department of Energy (Grant DE-FG02-97ER25308)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049

COLORECTAL CARCINOMA IN PATIENTS LESS THAN 40 YEARS OF AGE : PATHOLOGY AND PROGNOSIS

Clinicopathologic features in 35 patients below the age of 40 years with colorectal carcinoma were reviewed and compared with those of a control group containing 409 patients ranging from 40 to 74 years of age. All cases in this series were experienced at the First Department of Surgery, Nagasaki University School of Medicine, during the 18 years from 1970 to 1988. 1) Young patients accounted for 6.7% of all cases with colorectal cancer, and this group included more female patients than in the elderly control group. 2) The young patient group presented with advanced lesions (Dukes\u27 stage C) in 54.3% of cases compared with 35.6% of the old patient group with significantly high incidences of lymph node involvement. There were no significant differences in the hepatic metastasis and peritoneal dissemination among two groups. 3) Mucinous carcinoma and vascular invasion were frequently found on histologic examination in the young group, which suggested the highly malignant potentiality, but no significant difference in DNA ploidy pattern was observed between the young and the control group. 4) Curative resection rates were 71.4% in young and 68.1% in elderly patients. Overall 5-year survival was 56.9%. Five-year survival in 25 patients undergoing potentially curative resection increased to 74.4% with the similar rates of the control group. Improved 5-year survival following potentially curative resection stresses the need for early diagnosis and treatment

Tumor Markers (CEA, CA19-9, TPA) in Portal Blood in Colorectal Cancer Patients

The Carcinoembryonic antigen (CEA), CA19-9 and Tissue Polypeptide Antigen (TPA) levels in portal blood in colorectal cancer patients were studied in correlation with the peripheral blood levels and histopathologic findings in order to know serum levels increased. 1) Portal blood CEA, CA19-9 and TPA increased by operative maneuver. 2) Mean values of these markers in portal blood were higher than those in peripheral blood. 3) Portal blood CEA was correlated with Dukes\u27 staging, and revealed higher positive rates than CEA in peripheral blood in each stage. Portal CA19-9 changed within normal value and strikingly rose in Dukes\u27 D stage. Portal TPA tends to be higher in all stages and correlated with grades, but the value in Dukes\u27 D were lower than that of peripheral blood. 5) Moderately differentiated adenocarcinoma revealed the highest level of portal CEA (P<0.05), but portal CA19-9 and TPA did not indicate any correlation with cell differentiation. 6) The mean values of portal and peripheral CEA, CA19-9 and TPA showed significant elevation in those with infiltration of cancer cells extending through the proper muscle layer. This study suggests that the mechanism of these markers\u27 transfer from tumor into the portal vein is the most important decisive factor of the peripheral levels

Structure and Engineering of Francisella novicida Cas9

Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAM, thus expanding the target space of the CRISPR-Cas9 toolbox

Nuclear Pore Complex Protein Mediated Nuclear Localization of Dicer Protein in Human Cells

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein