The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.

Background: To examine if the significantly associated SNPs derived from the genome wide allelic association study on the AREDS cohort at the NEI (dbGAP) specifically confer risk for neovascular age-related macular degeneration (AMD). We ascertained 134 unrelated patients with AMD who had one sibling with an AREDS classification 1 or less and was past the age at which the affected sibling was diagnosed (268 subjects). Genotyping was performed by both direct sequencing and Sequenom iPLEX system technology. Single SNP analyses were conducted with McNemar's Test (both 2 × 2 and 3 × 3 tests) and likelihood ratio tests (LRT). Conditional logistic regression was used to determine significant gene-gene interactions. LRT was used to determine the best fit for each genotypic model tested (additive, dominant or recessive).

Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe a detailed protocol for the serial, hierarchical assembly of TALENs that requires neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. This restriction enzyme and ligation (REAL) protocol can be practiced using a small plasmid library and user-friendly, web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. All plasmids required to perform the REAL assembly method are publicly available through the Addgene plasmid distribution service (http://www.addgene.org/talengineering). Alternatively, to decrease the time and effort required, users can practice REAL using a library of plasmids encoding pre-assembled TALE repeats. With the platform of reagents, protocols, and software we describe, researchers can easily engineer multiple TALENs within two weeks or less using standard cloning techniques.

Purpose African Americans (AAs) have the highest incidence of colorectal cancer (CRC) compared to other US populations and more proximal CRCs. The objective is to elucidate the basis of these cancer disparities. . Experimental design 566 AA and 328 Non-Hispanic White (NHW) CRCs were ascertained in five Chicago hospitals. Clinical and exposure data were collected. Microsatellite instability and BRAF (V600E) and KRAS mutations were tested. Statistical significance of categorical variables was tested by Fisher's exact test or logistic regression and age by Mann-Whitney U test. Results Over a ten-year period, the median age at diagnosis significantly decreased for both AAs (68 to 61; P<0.01) and NHWs (64.5 to 62; P=0.04); more AA patients were diagnosed before age 50 than NHWs (22% vs 15%; P=0.01). AAs had more proximal CRC than NHWs (49.5% vs. 33.7%; P<0.01), but overall frequencies of microsatellite instability, BRAF and KRAS mutations were not different nor were they different by location in the colon. Proximal CRCs often presented with lymphocytic infiltrate (P<0.01) and were diagnosed at older ages (P=0.02). Smoking, drinking, and obesity were less common in this group, but results were not statistically significant. Conclusions Patients with CRC have gotten progressively younger. The excess of CRC in AAs predominantly consists of more proximal, microsatellite stable tumors, commonly presenting lymphocytic infiltrate and less often associated with toxic exposures or a higher BMI. Younger AAs had more distal CRCs than older ones. These data suggest two different mechanisms driving younger age and proximal location of CRCs in AAs.

VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) is essential for developmental and reparative angiogenesis. Reactive oxygen species (ROS) and copper (Cu) are also involved.in these processes. However, their inter-relationship is poorly understood. The role of endothelial Cu importer CTR1 in VEGFR2 signaling and angiogenesis in vivo is hitherto unknown. Here we show that CTR1 functions as a previously unrecognized redox sensor to promote angiogenesis in ECs. CTR1-depleted ECs showed reduced VEGF-induced VEGFR2 signaling and angiogenic responses. Mechanistically, CTR1 was rapidly sulfenylated at Cys189 in cytosolic C-terminus upon VEGF stimulation, which induced CTR1-VEGFR2 disulfide bond formation and their co-internalization to early endosomes, driving sustained VEGFR2 signaling. In vivo , EC-specific Ctr1-deficient mice or CRISPR/Cas9-generated “redox-dead” Cys to Ala Ctr1 knock-in mutant mice had impaired developmental and reparative angiogenesis. Thus, oxidation of CTR1 at Cys189 promotes VEGFR2 internalization and signaling to enhance angiogenesis. Our study uncovers an important mechanism for ROS sensing through CTR1 to drive neovascularization.

Many colorectal cancers (CRCs) that exhibit microsatellite instability (MSI) are not explained by MLH1 promoter methylation or germline mutations in mismatch repair (MMR) genes, which cause Lynch syndrome (LS). Instead, these Lynch-like syndrome (LLS) patients have somatic mutations in MMR genes. However, many of these patients are young and have relatives with cancer, suggesting a hereditary entity. We performed germline sequence analysis in LLS patients

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported. The combined analysis of SOFT-TEXT compared outcomes in 4,690 premenopausal women with estrogen/progesterone receptor–positive (ER/PgR+) early breast cancer randomly assigned to 5 years of exemestane + ovarian function suppression (OFS) versus tamoxifen + OFS. After a median follow-up of 9 years, exemestane + OFS significantly improved disease-free survival (DFS) and distant recurrence-free interval (DRFI), but not overall survival, compared with tamoxifen + OFS. We now report DFS, DRFI, and overall survival after a median follow-up of 13 years. In the intention-to-treat (ITT) population, the 12-year DFS (4.6% absolute improvement, hazard ratio [HR], 0.79; 95% CI, 0.70 to 0.90; P < .001) and DRFI (1.8% absolute improvement, HR, 0.83; 95% CI, 0.70 to 0.98; P = .03), but not overall survival (90.1% v 89.1%, HR, 0.93; 95% CI, 0.78 to 1.11), continued to be significantly improved for patients assigned exemestane + OFS over tamoxifen + OFS. Among patients with human epidermal growth factor receptor 2-negative tumors (86.0% of the ITT population), the absolute improvement in 12-year overall survival with exemestane + OFS was 2.0% (HR, 0.85; 95% CI, 0.70 to 1.04) and 3.3% in those who received chemotherapy (45.9% of the ITT population). Overall survival benefit was clinically significant in high-risk patients, eg, women age < 35 years (4.0%) and those with > 2 cm (4.5%) or grade 3 tumors (5.5%). These sustained reductions of the risk of recurrence with adjuvant exemestane + OFS, compared with tamoxifen + OFS, provide guidance for selecting patients for whom exemestane should be preferred over tamoxifen in the setting of OFS.

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double strand break (DSB), Cas9 remains stably bound to it. Here we show persistent Cas9 binding blocks access to DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches one direction towards the Cas9-DSB complex. By exploiting these RNA polymerase-Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of PAM sequences and a simple method of improving selection of highly active sgRNA for genome editing. STAR METHODS Contact for Reagent and Resource SharingFurther information and requests for reagents can be directed to, and will be fulfilled, by the corresponding author Bradley J. Merrill (merrillb@uic.edu). Experimental Model and Subject Details Cell cultureMouse embryonic stem (ES) cells harboring the Rex1:EGFPd2 insertion (A gift from Dr. Austin Smith) (24), or a Rosa26::TetOn-Otx2-mCherry insertion(A gift from Dr. Shen-his Yang) (25) were maintained 10cm dishes previously coated with 0.1% gelatin in Knockout DMEM media supplemented with the following: 15% KnockOut Serum Replacement, 2mM l-Glutamin, 1mM HEPES, 1× MEM NEAA, 55μM 2-mercaptoethanol, 100 U/ml LIF, and 3μM CHIR99021. Cell cultures were routinely split 1:10 with 0.25% trypsin-EDTA every 2-3 days. Method DetailsRecombinant Cas9 purification Cas9 (pMJ806, Addgene #39312) was expressed and purified by a combination of affinity, ion exchange and size exclusion chromatographic steps as previously described (20).