GO: a functional reporter system to identify and enrich base editing activity.

Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a 'Gene On' (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes or enzymes, such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.

Germline competency of human embryonic stem cells depends on eomesodermin.

In humans, germline competency and the specification of primordial germ cells (PGCs) are thought to occur in a restricted developmental window during early embryogenesis. Despite the importance of specifying the appropriate number of PGCs for human reproduction, the molecular mechanisms governing PGC formation remain largely unexplored. Here, we compared PGC-like cell (PGCLC) differentiation from 18 independently derived human embryonic stem cell (hESC) lines, and discovered that the expression of primitive streak genes were positively associated with hESC germline competency. Furthermore, we show that chemical inhibition of TGFß and WNT signaling, which are required for primitive streak formation and CRISPR/Cas9 deletion of Eomesodermin (EOMES), significantly impacts PGCLC differentiation from hESCs. Taken together, our results suggest that human PGC formation involves signaling and transcriptional programs associated with somatic germ layer induction and expression of EOMES.

Generation of precision preclinical cancer models using regulated in vivo base editing.

Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.

Tankyrase inhibition sensitizes cells to CDK4 blockade.

Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the ß-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.

The TFAP2C-Regulated OCT4 Naive Enhancer Is Involved in Human Germline Formation.

Human primordial germ cells (hPGCs) are the first embryonic progenitors in the germ cell lineage, yet the molecular mechanisms required for hPGC formation are not well characterized. To identify regulatory regions in hPGC development, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to systematically characterize regions of open chromatin in hPGCs and hPGC-like cells (hPGCLCs) differentiated from human embryonic stem cells (hESCs). We discovered regions of open chromatin unique to hPGCs and hPGCLCs that significantly overlap with TFAP2C-bound enhancers identified in the naive ground state of pluripotency. Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity.

The effect of micronized estradiol on bone turnover and calciotropic hormones in older men receiving hormonal suppression therapy for prostate cancer.

To examine the effect of estradiol (E(2)) without the confounding effect of hypothalamic-pituitary feedback, we studied men with prostate cancer in whom gonadotropin secretion was suppressed by LH-releasing hormone agonists (LHRH-A). Fourteen men over 65 yr of age and receiving established LHRH-A treatment (EST group) without bony metastases and 12 men who received LHRH-A as neoadjuvant therapy for locally advanced prostate cancer (NEO group) were randomized (double blind) to receive either 1 mg/d micronized E(2) (n = 12) or placebo (PL; n = 13) for 9 wk. E(2), estrone, testosterone, SHBG, PTH, and 25-hydroxy- and 1,25-dihydroxyvitamin D levels as well as markers of bone resorption [N- and C-telopeptide cross-links (NTX and CTX) and deoxypyridinoline] and bone formation (bone-specific alkaline phosphatase, osteocalcin, and N-terminal type I collagen) were measured before LHRH-A in the NEO group, before [baseline (BL)] and after 9 wk of E(2) or PL in all patients, and 6 wk after E(2) treatment in the EST group. In the NEO group, hormone levels fell 3 wk after the initial LHRH-A injection, and deoxypyridinoline increased significantly (P = 0.006). At BL, the EST group had higher bone turnover due to the longer duration of LHRH-A treatment. With E(2) treatment, E(2) levels rose into the normal male range, and two resorption markers decreased significantly from BL by 33% for NTX (P < 0.001) and 28% for CTX (P = 0.009). Bone formation markers did not change. PTH increased by 43% from BL (P < 0.01) in the E(2) group and decreased 16% from BL in the PL group (P < 0.01). Ionized calcium did not change in the E(2) group, but increased in the PL group by 2.3% (P < 0.01). NTX and CTX increased 6 wk after E(2) withdrawal in the EST group. We conclude that E(2) inhibits bone resorption in hypogonadal men through a direct skeletal effect that is independent of PTH. Low dose estrogen may be an option for the prevention and/or treatment of bone loss in this population.

The effect of short-term estradiol therapy on cognitive function in older men receiving hormonal suppression therapy for prostate cancer.

OBJECTIVES: To determine the effect of estrogen (E) alone (without the influence of testosterone (T)) on cognitive function in older men, using 17-beta micronized estradiol versus placebo in older men rendered hypogonadal (low T and E) by treatment for prostate cancer. DESIGN: Short-term double-blind, randomized, controlled trial. SETTING: An outpatient General Clinical Research Center. PARTICIPANTS: Twenty-seven community-dwelling men aged 65 and older receiving neoadjuvant or established therapy with luteinizing-hormone releasing-hormone agonists for treatment of prostate cancer enrolled in a short-term randomized, controlled trial of 17-beta micronized estradiol versus placebo on the effect on biochemical markers of bone turnover. MEASUREMENTS: Hormone levels, including E, T, and sex hormone-binding globulin; standardized neurocognitive tests, including measures of sustained attention, executive function, and memory; and questionnaires to assess subjects' perception of cognitive deficits and symptoms of depression. RESULTS: There were no significant differences between patients receiving E or placebo on 15 of 17 neurocognitive measures and no significant differences in self-reported cognitive deficits or number of depressive symptoms. CONCLUSION: Although studies have suggested that E replacement therapy may improve cognitive function, most notably memory performance in postmenopausal woman, there was no evidence in the present study that the addition of short-term E therapy was more beneficial than placebo in tests of cognitive performance in hypogonadal men.

Medication reconciliation at an academic medical center: implementation of a comprehensive program from admission to discharge.

PURPOSE: The implementation of a comprehensive medication reconciliation program to reduce errors in admission and discharge medication orders at an academic medical center is described. SUMMARY: A multidisciplinary team was formed to assess the current process of obtaining medication histories and to develop a new workflow for the pharmacist to obtain and reconcile medication histories. Pharmacists received intensive training on the new workflow, policies, and procedures. Hospitalwide multidisciplinary education was provided, and the new process was introduced in November 2005. Every inpatient admitted to the hospital has a complete and comprehensive home medication history interview conducted by a pharmacist or designee (pharmacy student or intern with subsequent verification by a pharmacist) within 24 hours of arrival. All components of the medication history are documented utilizing an integrated electronic medical record (EMR) medication documentation tool. Development of the discharge medication reconciliation program began in fall 2006. A discharge medication reconciliation report form was created through the EMR to improve the accuracy of the discharge medication orders. The form provides physicians with complete, accurate medication information and decreases the risk for transcription errors. Finally, a discharge medication report was developed for patients to take home. Analysis of the discharge reconciliation process revealed that medication errors were reduced from 90% to 47% on the surgical unit (95% confidence interval [CI], 42-53%; p = 0.000) and from 57% to 33% on the medicine unit (95% CI, 28-38%; p = 0.000). CONCLUSION: A pharmacy-driven multidisciplinary admission history and medication reconciliation process has reduced medication errors in an academic medical center.