Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out.

Hao Zhang,Zihao Shen,Huajun Sheng,Shujuan Zhu,Zhen Guo,Ziling Cai,Qingqian Xia,Yu Xing,Qian Liu,Zhi-Yao He

doi:10.1371/journal.pone.0263162

Abstract

Among the three existing targeted gene editing technologies, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9), the latter is widely used owing to its simplicity, efficiency, and low cost. Here, we routinely infected A172 and U251 cells with lentiviral vectors, in which aquaporin-8 (AQP8) was knocked out using CRISPR/Cas9. Our results indicated that cryopreservation did not significantly alter the viral infection efficiency, but influenced AQP8 expression in the infected cells at both protein and mRNA levels compared with the non-cryopreserved samples. Further, AQP8 expression at protein and mRNA levels in recovered cryopreserved infected cells did not significantly differ from those in the blank and negative controls, indicating that the lentivirus was still infectious at low temperatures. However, it failed to release the AQP8-targeting guide RNA in the infected cells, or the guide RNA was released, but underwent changes that caused it to malfunction in the cells with CRISPR/Cas9-mediated AQP8 knock-out. Our findings possibly provide some insights into the reliability of lentiviruses as CRISPR/Cas9 vectors.

Highlights

The clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR/ Cas9) gene editing technology, which is widely used, is a convenient and efficient strategy for precisely and site- modifying specific gene sequences of interest [1–3]
We counted cells with and without cryopreservation in bright and fluorescent fields, and thereafter determined the degree of viral infection in the A172 and U251 cells using the fluorescent to bright field data ratio (Table 1)
The CRISPR/Cas9 gene editing technology was used to construct two AQP8 knockout viruses, GV371 (U6-sgRNA-SV40-EGFP) and Lenti-cas9-puro (Puro represents puromycin used for screening uninfected cells)

Summary

Introduction

The clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR/ Cas9) gene editing technology, which is widely used, is a convenient and efficient strategy for precisely and site- modifying specific gene sequences of interest [1–3]. Cryopreservation is a means by which cells can be temporarily stored at low temperatures to prevent sustained growth, and reportedly, various types of cells transfected with specific plasmids or infected with specific viruses can be cryopreserved for later use [4]. The initial objective of this study was to infect glioma cell lines with AQP8 knock out so as to explore the effect of AQP8 on their proliferation. We constructed an aquaporin-8 (AQP8) knock-out viral. Construction of an aquaporin-8 (AQP8) knock-out viral vector used to infect A172 and U251 cells vector using CRISPR/Cas and thereafter, used it to infect A172 and U251 cells. We incidentally observed that cryopreservation was unfavorable for infected cells with inhibited AQP8 expression; it might not be applicable to all cell types

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Conclusion