Transcriptional Inhibition of lncRNA gadd7 by CRISPR/dCas9-KRAB Protects Spermatocyte Viability.

Jun Zhao,Yucheng Zhong,Meiqiong Yang,Wenmin Ma,Yaqin Wu,Huan Li,Hao Deng,Bingyu Zhou

doi:10.3389/fmolb.2021.652392

Jun Zhao, Yucheng Zhong + Show 6 more

Open Access

https://doi.org/10.3389/fmolb.2021.652392

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Abstract

Our previous study found that lncRNA gadd7 was up-regulated in the semen of varicocele patients, and could promote the apoptosis of mouse spermatocytes and inhibit their proliferation. Therefore, we further explored whether down-regulation of Gadd seven expression could protect the viability of spermatocytes. Here we designed specific sgRNAs targeting the ORF region of gadd7, and constructed a CRISPR-dCas9-KRAB system that effectively inhibits gadd7 at the transcriptional level. The CRISPRi system can effectively prevent the apoptosis of spermatocytes and enhance their proliferation, which is expected to provide a potentially effective molecular intervention method for the treatment of male infertility caused by varicocele.

Highlights

Varicocele is a common disease of the male reproductive system (Hyun, 2020)
We designed a specific sgRNA for the ORF region of gadd7, and screened and obtained a CRISPRi system that effectively inhibits gadd7. This system can effectively slow down the apoptosis of spermatocytes and promote their proliferation, which is expected to provide a potentially effective molecular intervention method for the treatment of male infertility caused by varicocele
To determine whether the CRISPRi technology can be used to inhibit the expression of lncRNA gadd7, we targeted the dCas9KRAB protein to the ORF regions of gadd7 using designed sgRNAs (Figure 1A)

Summary

INTRODUCTION

Varicocele is a common disease of the male reproductive system (Hyun, 2020). The incidence rate in the general male population is 15%, and the incidence rate in the infertile population is as high as 39% (Alsaikhan et al, 2016; Barratt et al, 2017). It is considered by the World Health Organization to be one of the most common causes of male infertility It affects the spermatogenic function of the testis through a variety of pathophysiological changes. Long noncoding RNAs (LncRNAs) are a type of RNA molecules with a transcript length of more than 200 nt (Kopp and Mendell, 2018) They are widely present in mammals and participate in various normal activities of cells, such as genome imprinting, transcription regulation, X Chromosome silencing and nuclear transport (Chen, 2016). We designed a specific sgRNA for the ORF region of gadd, and screened and obtained a CRISPRi system that effectively inhibits gadd7 This system can effectively slow down the apoptosis of spermatocytes and promote their proliferation, which is FIGURE 2 | Inactivation of gadd by CRISPR-dCas9-KRAB prevents cell apoptosis. Expected to provide a potentially effective molecular intervention method for the treatment of male infertility caused by varicocele

MATERIALS AND METHODS

RESULTS

DISCUSSION