11 research outputs found
Sexual dysfunction prevalence, risk factors, and help-seeking behavior in opioid agonist treatment and general psychiatry: a cross-sectional study
BackgroundMental disorders pose a high risk for the occurrence of sexual dysfunctions (SD). This study aimed to investigate prevalence of risk factors and help-seeking behavior for sexual dysfunctions in patients with opioid use disorder compared to patients seeking psychotherapeutic help.MethodsNinety-seven patients at two opioid agonist treatment (OAT) centers and 65 psychotherapeutic patients from a psychiatric practice (PP) in Switzerland were included in the study. Self-report assessments comprised sexual functioning (IIEF: International Index of Erectile Function; FSFI: Female Sexual Function Index), depressive state, psychological distress, alcohol consumption, nicotine use, and a self-designed questionnaire on help-seeking behavior. We used chi-squared and Mann–Whitney U tests for group comparisons and binary logistic regression models to identify variables predicting the occurrence of sexual dysfunctions.ResultsThere was no statistically significant difference (p = 0.140) in the prevalence of SD between OAT (n = 64, 66.0%) and PP sample (n = 35, 53.8%). OAT patients scored significantly higher in scales assessing nicotine use (p < 0.001) and depressive state (p = 0.005). Male OAT patients scored significantly worse on the Erectile Function scale (p = 0.005) and female PP patients scored significantly worse on the FSFI Pain domain (p = 0.022). Opioid use disorder, higher age, and being female predicted the occurrence of SD in the total sample. In the OAT sample, only higher age remained predictive for the occurrence of SD. A lack of help-seeking behavior was observed in both groups, with only 31% of OAT patients and 35% of PP patients ever having talked about their sexual health with their treating physician.ConclusionSD are common among psychiatric patients receiving OAT and general psychiatric patients seeking psychotherapy. Professionals providing mental healthcare to patients must emphasize prevention and routine assessments of sexual functioning needs
Targeted removal of epigenetic barriers during transcriptional reprogramming
Master transcription factors dominantly direct cell fate and barriers ensuring their tissue specific silencing are not clearly defined. Here, the authors demonstrate that inefficient targeted transactivation of Sox1 in neural progenitor cells is surmountable through targeted promoter demethylation using dCas9-Tet1
One step generation of customizable gRNA vectors for multiplex CRISPR approaches through string assembly gRNA cloning (STAgR).
Novel applications based on the bacterial CRISPR system make genetic, genomic, transcriptional and epigenomic engineering widely accessible for the first time. A significant advantage of CRISPR over previous methods is its tremendous adaptability due to its bipartite nature. Cas9 or its engineered variants define the molecular effect, while short gRNAs determine the targeting sites. A majority of CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into single cells, either as an essential precondition, to increase responsive cell populations or to enhance phenotypic outcomes. Despite these requirements, methods allowing the efficient generation and delivery of multiple gRNA expression units into single cells are still sparse. Here we present STAgR (String assembly gRNA cloning), a single step gRNA multiplexing system, that obtains its advantages by employing the N20 targeting sequences as necessary homologies for Gibson assembly. We show that STAgR allows reliable and cost-effective generation of vectors with high numbers of gRNAs enabling multiplexed CRISPR approaches. Moreover, STAgR is easily customizable, as vector backbones as well as gRNA structures, numbers and promoters can be freely chosen and combined. Finally, we demonstrate STAgR's widespread functionality, its efficiency in multi-targeting approaches, using it for both, genome and transcriptome editing, as well as applying it in vitro and in vivo
CORALINA. A universal method for the generation of gRNA libraries for CRISPR-based screening
Köferle A, Worf K, Breunig C, et al. CORALINA. A universal method for the generation of gRNA libraries for CRISPR-based screening. BMC Genomics. 2016;17(1): 917.Background
The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches.
Results
Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs.
Conclusions
The simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens
CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening
Application of STAgR.
<p>(A) Colony PCR of a 6xSTAgR reaction using two different promoters as well as both, the canonical and the SAM loop gRNA scaffold. The gel shows a colony PCR of 22 bacterial colonies, of which seven showed the amplicon indicative of the full length STAgR reaction (2444bp). (B) Exemplary colony PCR of STAgR constructs with 0 to 8 gRNA expression cassettes. (C) A STAgR plasmid containing four gRNAs or a mixture of four single gRNA plasmids have been transfected into P19 Cells expressing dCas9-VPR. (D) After 7 days mRNA was extracted and transcript levels of target genes have been compared via qPCR. Error bars depict standard errors of the mean.</p
Functional validation of STAgR.
<p>(A) Colony PCR of a 4xSTAgR reaction (using a string sequence containing a hU6 promoter and a canonical gRNA scaffold). 24 bacterial colonies are shown, of which six present the amplicon size indicative of the full length reaction (1596 bp). Additionally marked are amplicon sizes indicative of two (823 bp) and single gRNAs (458 bp). (B) Quantification of cloning efficiencies from three different 4xSTAgR reactions (n = 130). (C) A schematic showing constructs used for functional validation of STAgR gRNAs. A gRNA targeting the GFP ORF was either delivered in a single gRNA expression vector or on each of four different positions in STAgR vectors. (D) Functional validation of STAgR vectors shown in Fig 2C. HeLa cells stably expressing d2GFP and Cas9 have been transfected with vectors depicted above. Flow cytometry indicates that STAgR constructs are similarly efficient in mutating the ORF of GFP compared to a single gRNA vector. (E) Colony PCR of a 4xSTAgR reaction using four different promoters and SAM loop scaffolds. 24 bacterial colonies are shown, of which seven colonies incorporated the amplicon size indicative of the full length reaction (2043 bp). Shorter amplicons are indicative of gRNA subsets, which vary in size, depending on the incorporated promoter.</p
The STAgR protocol.
<p>(A) An Overview over STAgR procedure. STAgR allows simple and fast generation of multiplexing vectors in one overnight reaction. STAgR is also highly customizable as diverse strings and vectors can be used to assemble expression cassettes with different promoters and gRNA scaffolds. (B) Sequences of overhang primers used for generation of STAgR vectors.</p
Additional file 7: Figure S3. of CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening
Analysis of CORALINA libraries. Bowtie has been used to classify targeting sites of gRNAs derived from human (L2, A or L3, B) CORALINA libraries. Pie charts indicating relative proportion of functional domains bound by gRNAs (middle). gRNA protospacer aligning to coding gene units (left) or repeats (right) are further sub-classified. Promoters are defined here as genomic sequences 10 kb upstream the transcriptional start site. Coding and noncoding gene and repeat information has been derived from UCSC. Numbers next to the sectors depict the median number of genomic alignments for the selected group of gRNAs. (PDF 73 kb
Additional file 4: Figure S4. of CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening
Functional analysis of CORALINA gRNAs. (A) List of most frequently sequenced alteration generated with gRNA P1-20. (B-E) Top: Schematic depicting CORALINA-derived gRNAs targeting regions in or near various human genes (HS3ST3B1 gRNA H1-46, 46 bp protospacer, PCDH8, gRNA P2- 40, 40 bp protospacer, ZNF790, gRNA Z1-35, 35 bp protospacer, PIK3AP1, gRNA P3-35, 35 bp protospacer). Control gRNAs have been shortened from the 5′ end to yield a 20 bp protospacer. Right: Bargraph depicting percentage of NGS reads displaying indels after targeting wild-type Cas9 using CORALINA-derived gRNAs in HEK293T cells. Below: List of the most frequently sequenced alterations generated by CORALINA and control gRNAs. (PDF 320 kb