Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs.

BACKGROUND: The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed. One such method is Restriction Enzyme (RE) mapping. Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously. METHODS: In this study, the sgRNA templates were initially cloned into the above PX459 plasmids. Subsequently, the accuracy of the constructs was determined by RE mapping. RESULTS: This method was established to screen for sgRNA-bearing PX459 plasmids. However, numerous anomalies were detected after ligation of sgRNA templates into RE digested PX459 plasmids. CONCLUSION: Our data suggest that RE mapping is only appropriate as an initial screen and that the identity of all plasmids with the correctly identified RE maps should be confirmed by Sanger sequencing.

Association between mitochondrial DNA content and opium exposure.

To date, not much study has been done to investigate the mitochondrial DNA (mtDNA) copy number as the potential biomarker for opium exposure. Here, we conducted a cross-sectional study to determine the relative mtDNA content as the potential biomarker for opium exposure. Quantitative real-time PCR was performed to investigate the mtDNA copy number variation across 205 individuals, including blood samples of 45 opium users, 41 cigarette users, 47 dual users, and 72 never users of any product. We found a significantly higher mtDNA content among the opium-only users (adjusted OR: 3.21; 95% CI: [1.34, 7.66]; P = .009) and dual users (adjusted OR: 2.64; 95% CI: [1.15, 6.1]; P = .02) compared to that in never users even after adjustment for confounding factors, age, and sex. Discordantly, analysis of mitochondrial DNA in cigarette smokers revealed an indirect association between cigarette smoking and mtDNA content although it was not statistically significant. The reason behind the increased mitochondrial DNA is unclear. The possible hypothesis is that there might be a way to compensate for the oxidative damage induced by opium consumption. Taken together, our findings indicated that the mtDNA copy number may alter during opium exposure. Since changes in the mitochondrial DNA copy number was associated with the etiology of many diseases including cancer, further investigations on the mtDNA copy number may shed light on the carcinogenicity of opium consumption and means for early detection among the populations who have been exposed to opium and its products.

Expression and Clinicopathological Significances of lncRNAs: Could ARA and ZEB2NAT be the Potential Breast Cancer-Related Biomarkers?

INTRODUCTION: Pieces of evidence have shown that a significant proportion of cancer-prone factors are not attributed to alterations in protein-coding sequences. Adriamycin resistance-related (ARA) and natural antisense of ZEB2 (ZEB2NAT) long non-coding RNAs (lncRNAs) have been indicated with oncogenic properties by regulating various signaling pathways and epithelial-to-mesenchymal transition (EMT), which may have diagnostic and prognostic potential as a novel group of biomarkers. AIM: The current study aimed to evaluate the expression status of ARA and ZEB2NAT lncRNAs and their clinicopathological significance in a population with breast cancer (BC). METHODS: Total RNA was extracted from 60 tumor samples and their normal adjacent tissues (NATs). The lncRNA expressions were measured using quantitative reverse transcription PCR (RT-qPCR) and statistical analyses were performed by SPSS version 25. RESULTS: Our data showed a significant upregulation of ARA and ZEB2NAT lncRNAs in tumor tissues compared to NATs (p <0.001; p = 0.021, respectively). ARA and ZEB2NAT expression were observed to be significantly associated with tumor grade, nuclear grade, tumor stages, and lymph node metastasis (p <0.05). Additionally, ARA expression was significantly correlated with breastfeeding status (p = 0.027). CONCLUSION: our data revealed that ARA and ZEB2NAT lncRNAs were overexpressed in BC. Furthermore, the selected lncRNAs were found to might be the potential biomarkers for BC diagnosis and prognosis. However, the findings of the current research are required to be replicated in other studies with larger sample sizes.

Expression and hydroxyurea-triggered induction of EGFP upon CRISPR/Cas9-mediated integration into the γ-globin gene of K562 cells.

OBJECTIVE: To knock-in an EGFP cassette into the γ-globin genes of K562 cells via CRISPR/Cas9, and to assess expression and hydroxyurea (HU)-mediated induction of the targeted EGFP transgene. RESULTS: The EGFP cassettes were specifically knocked into the Gγ gene. EGFP expression was detected in the targeted cell population and isolated clones. Furthermore, EGFP transcript and fluorescence levels were significantly induced following HU-treatment. CONCLUSION: This system is readily utilizable for genome scale studies of cis-acting regulatory elements which are implicated in γ-globin expression or HU-mediated induction.