Establishment of an miR‑137‑knockout cell model using CRISPR/Cas9 genome editing

Chen,Wei; Li,Qi; Du,Jingjie; Li,Xiaodi; Jiang,Songshan; He,Yuanli

doi:10.3892/ol.2018.9096

Establishment of an miR‑137‑knockout cell model using CRISPR/Cas9 genome editing

Authors:
- Wei Chen
- Qi Li
- Jingjie Du
- Xiaodi Li
- Songshan Jiang
- Yuanli He
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Affiliations: Department of Gynecology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510260, P.R. China, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat‑sen University, Guangzhou, Guangdong 510006, P.R. China, Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510260, P.R. China
Published online on: July 6, 2018 https://doi.org/10.3892/ol.2018.9096
Pages: 4027-4032

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Abstract

MicroRNA‑137 (miR‑137) has been reported to be abnormally expressed in a variety of types of cancer, including ovarian cancer. However, the roles served by miR‑137 in cancer are not fully understood. In the present study, 3 single guide RNAs (sgRNAs) were designed, synthesized and inserted into pXPR001 plasmids. The pXPR001‑sgRNA plasmids were verified using sequencing and integrated into the genome of the ovarian cancer cell line, A2780, through lentiviral transduction, puromycin selection and single‑cell culture. PCR products amplified from single‑cell cultures using primers covering miR‑137 targeting sites were sequenced to identify clones with miR‑137 gene disruption. Genome editing was detected in 72% of the clones derived from sgRNA2, 4% from sgRNA3 and 0% from sgRNA1. Of the clones from sgRNA2, 32% contained 1 edited miR‑137 allele and 40% contained 2 edited miR‑137 alleles. The expression of miR‑137 in clones #2 and #3 could not be detected by reverse transcription‑quantitative polymerase chain reaction. In addition, an MTT assay demonstrated that clones #2 and #3 exhibited enhanced proliferation. In conclusion, an miR‑137‑knockout cell model was successfully established in A2780 cells using CRISPR/Cas9 technology.

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