Adrenocortical carcinoma (ACC) is an aggressive tumor showing frequent metastatic spread and poor survival. Although recent genome-wide studies of ACC have contributed to our understanding of the disease, major challenges remain for both diagnostic and prognostic assessments. The aim of this study was to identify specific microRNAs (miRNAs) associated with malignancy and survival of ACC patients. miRNA expression profiles were determined in a series of ACC, adenoma, and normal cortices using microarray. A subset of miRNAs showed distinct expression patterns in the ACC compared with adrenal cortices and adenomas. Among others, miR-483-3p, miR-483-5p, miR-210, and miR-21 were found overexpressed, while miR-195, miR-497, and miR-1974 were underexpressed in ACC. Inhibition of miR-483-3p or miR-483-5p and overexpression of miR-195 or miR-497 reduced cell proliferation in human NCI-H295R ACC cells. In addition, downregulation of miR-483-3p, but not miR-483-5p, and increased expression of miR-195 or miR-497 led to significant induction of cell death. Protein expression of p53 upregulated modulator of apoptosis (PUMA), a potential target of miR-483-3p, was significantly decreased in ACC, and inversely correlated with miR-483-3p expression. In addition, high expression of miR-503, miR-1202, and miR-1275 were found significantly associated with shorter overall survival among patients with ACC (P values: 0.006, 0.005, and 0.042 respectively). In summary, we identified additional miRNAs associated with ACC, elucidated the functional role of four miRNAs in the pathogenesis of ACC cells, demonstrated the potential involvement of the pro-apoptotic factor PUMA (a miR-483-3p target) in adrenocortical tumors, and found novel miRNAs associated with survival in ACC.

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications but identifying unwanted off-target mutations is important for clinical translation. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.

21Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a 22 target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay 23 for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach 24 takes advantage of the recruitment of endogenous DNA repair factors for genome-wide 25 identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely 26 to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base 27 resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene 28 editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene 29 editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides 30 an unprecedented view of events that precede repair of the affected sites. 31 strengths, they also have certain weaknesses. Naïve prediction algorithms are for the most part 41 based on sequence similarity and currently have limited predictive power with very high false-42 positive rates (10). Assays that induce DSBs in vitro, such as Digenome-Seq (5), CIRCLE-Seq (6) 43 and SITE-Seq (7), have high sensitivity but dramatically under-or overestimate the number of 44 target sites that are actually modified in cellular models or in vivo (11). Nuclease concentration 45 within the cell (7), delivery method (ribonucleoprotein (RNP) vs. plasmid) (7, 12, 13) as well as 46 more complex cellular properties such as chromatin accessibility (14, 15) have been shown to 47

Spotting off-targets from gene editing Unintended genomic modifications limit the potential therapeutic use of gene-editing tools. Available methods to find off-targets generally do not work in vivo or detect single-nucleotide changes. Three papers in this issue report new methods for monitoring gene-editing tools in vivo (see the Perspective by Kempton and Qi). Wienert et al. followed the recruitment of a DNA repair protein to DNA breaks induced by CRISPR-Cas9, enabling unbiased detection of off-target editing in cellular and animal models. Zuo et al. identified off-targets without the interference of natural genetic heterogeneity by injecting base editors into one blastomere of a two-cell mouse embryo and leaving the other genetically identical blastomere unedited. Jin et al. performed whole-genome sequencing on individual, genome-edited rice plants to identify unintended mutations. Cytosine, but not adenine, base editors induced numerous single-nucleotide variants in both mouse and rice. Science , this issue p. 286 , p. 289 , p. 292 ; see also p. 234

Abstract. Colorectal cancer (CRC) is one of the most common and deadly forms of cancer. Despite improved treatment modalities, post-operative recurrence and metastasis remain the major problems for extending patient survival after surgery. This highlights the need to search for biomarkers for prognostication and treatment stratification of colorectal cancer patients. In this study, we applied the SYBR-green quantitative PCR-based array approach to screen for differentially expressed miRNAs between patients with short (<50 months, range 10-33 months) and long survival (≥50 months, range 50-152 months). The selected candidate prognostic miRNAs were validated in a cohort of 50 CRC patients by TaqMan quantitative PCR. We found that high expression of miR-185 and low expression of miR-133b were correlated with poor survival (p=0.001 and 0.028, respectively) and metastasis (p=0.007 and 0.036, respectively) in colorectal cancer. Our findings suggest the potential prognostic values of these miRNAs for predicting clinical outcome after surgery. IntroductionColorectal cancer (CRC) is the second most common cancer in women and the third in men worldwide (1). It ranks the second most common cause of cancer death in the Western world (2). Currently, there are several treatment modalities for CRC, including surgery, radiotherapy, chemotherapy and targeted therapy (e.g., cetuximab). However, the long-term survival remains low in metastatic disease (3).Given that CRC usually follows a stepwise progression from benign to malignant lesion and distant metastasis, there is a possibility for early diagnosis in order to reduce morbidity and mortality. The commonly used method to characterize CRC tumor in clinic is T1-T3 staging system. However, the staging system reflects only morphological characteristics of the tumor and does not consider tumor molecular biology, thus, it is inaccurate in predicting the future outcome for each particular case. Therefore, the development of screening tools and new biomarkers to facilitate early diagnosis is particularly warranted. Further investigations in the search for new prognostic biomarkers may help to improve post-operative treatment approaches for CRC patients.Several studies have documented a link between the aberrant expression of a class of small non-coding RNAs, termed microRNAs (miRNAs), and the pathogenesis/prognosis of several cancer types, including colorectal cancer (4,5). These molecules provide a potentially valuable diagnostic/prognostic tool for CRC pathology, because mature miRNA species are relatively more stable than mRNAs and well preserved in formalin-fixed, paraffin-embedded samples which are commonly used in clinical routine (6). Furthermore, only a small number of miRNAs are required to distinguish cancerous tissues from non-cancerous tissues compared with mRNA profiles (7), which makes them more feasible candidate biomarkers.miRNAs are endogenous single-stranded non-coding RNAs of ~22-nucleotides in length, which are generated by an RNase III enzyme Dicer from endogenou...