No abstract

Genome editing reemerged in 2012 with the development of CRISPR/Cas9 technology, which is a genetic manipulation tool derived from the defense system of certain bacteria against viruses and plasmids. This method is easy to apply and has been used in a wide variety of experimental models, including cell lines, laboratory animals, plants, and even in human clinical trials. The CRISPR/Cas9 system consists of directing the Cas9 nuclease to create a site-directed double-strand DNA break using a small RNA molecule as a guide. A process that allows a permanent modification of the genomic target sequence can repair the damage caused to DNA. In the present study, the basic principles of the CRISPR/Cas9 system are reviewed, as well as the strategies and modifications of the enzyme Cas9 to eliminate the off-target cuts, and the different applications of CRISPR/Cas9 as a system for visualization and gene expression activation or suppression. In addition, the review emphasizes on the potential application of this system in the treatment of different diseases, such as pulmonary, gastrointestinal, hematologic, immune system, viral, autoimmune and inflammatory diseases, and cancer.

Persistent high-risk human papillomavirus (HR-HPV) infections play a major role in the development of invasive cervical cancer (CC), and screening for such infections is in many countries the primary method of detecting and preventing CC. HPV typing can be used for triage and risk stratification of women with atypical squamous cells of undetermined significance (ASC-US)/low-grade cervical lesions (LSIL), though the current clinical practice in Mexico is to diagnose CC or its preceding conditions mainly via histology and HR-HPV detection. Additional information regarding these HPV infections, such as viral load and co-infecting agents, might also be useful for diagnosing, predicting, and evaluating the possible consequences of the infection and of its prevention by vaccination. The goal of this follow-up hospital case study was to determine if HPV types, multiple HPV infections, and viral loads were associated with infection persistence and the cervical lesion grade. A total of 294 cervical cytology samples drawn from patients with gynecological alterations were used in this study. HPV types were identified by real-time PCR DNA analysis. A subset of HPV-positive patients was reevaluated to identify persistent infections. We identified HPV types 16, 18, and 39 as the most prevalent. One hundred five of the patients (59%) were infected with more than one type of HPV. The types of HPV associated with multiple HPV infections were 16, 18, and 39. In the follow-up samples, 38% of patients had not cleared the initially detected HPV infection, and these were considered persistent. We found here an association between multiple HPV infections and high viral loads with and infection persistence. Our findings suggest there are benefits in ascertaining viral load and multiple HPV infections status of HR-HPV infections for predicting the risk of persistence, a requirement for developing CC. These findings contribute to our understanding of HPV epidemiology and may allow screening programs to better assess the cancer-developing risks associated with individual HR-HPV infections.

Recombinants in which the six GC-motifs (I -VI) present in the upstream element of the SV40 early promoter region have been point mutated either individually or in pairs were used to determine the possible contribution of each GC-motif to the function of the overlapping early-early and late-early SV40 promoters. GC-motif I, and to a lesser extent, GC-motifs H and HI, are critical for initiation at the early-early start sites. GC-motifs IV -VI play a subsidiary role. Mutations in GCmotifs I and II do not decrease the activity of the late-early promoter, whereas mutations in the GC-motifs m -VI have a moderate effect on it. The in vivo phenotype of the GCmotif mutants can be almost fully reproduced in vitro using a nuclear extract. DNase I protection footprinting experiments using wild-type or mutated templates and nuclear extracts indicate that each GC-motif behaves principally as an independent protein-binding site, presumably for transcription factor Spl. The effect of changing the position of the 21-bp repeat region on initiation from the early-early and late-early start sites indicates that there is little flexibility in the position in which this upstream element can efficiently activate initiation of transcription from these start sites.Key words: RNA polymerase B(LI) promoter/upstream element/in vivo, in vitro transcription/transcription factor/DNase I footprinting.

The role of the 21-bp repeat region [simian virus 40 (SV40) coordinates 40-103] on early and late SV40 promoter functions has been investigated in vitro using a variety of mutated templates. Using either a HeLa whole cell extract or a S100 extract, we analyzed the transcripts by quantitative Si nuclease mapping. GC-rich motifs contained in the 21-bp direct repeat constituted an essential element for efficient early transcription in vitro in agreement with previous in vivo results. These GC-rich motifs act in a non-polar fashion, since inversion of the 21-bp region did not reduce early transcription. Some point mutations in the 22-bp imperfectly repeated sequence, that drastically reduce initiations from the early promoter in vivo, had little effect in vitro, indicating that all the functions of these GC-rich motifs cannot be reproduced in vitro at present. The requirement for the 21-bp repeat region was less stringent when the concentration of the early promoter sequence was increased, which suggests that its function may be to facilitate the recognition of the 'weak' SV40 early TATA box. The multiple late start sites were accurately used in vitro and the GC-rich motifs contained in the 21-bp repeat region were an important element for efficient in vitro initiation of transcription from the late promoter, irrespective of their orientation. However, the effect of the 21-bp repeat region on late initiations decreased strikingly with increasing distance to the start sites, although it was still detectable over a distance of 220 bp. Under the present in vitro conditions, the 72-bp repeat region stimulates weakly both early and late transcription.