Targeting cellular mRNAs translation by CRISPR-Cas9.

Yuchen Liu,Hanwei Wu,Weiren Huang,Junhao Lin,Li Liu,Anbang He,Qiaoxia Zhang,Jianfa Li,Chengle Zhuang,Zhicong Chen,Yonghao Zhan

doi:10.1038/srep29652

Abstract

Recently CRISPR-Cas9 system has been reported to be capable of targeting a viral RNA, and this phenomenon thus raises an interesting question of whether Cas9 can also influence translation of cellular mRNAs. Here, we show that both natural and catalytically dead Cas9 can repress mRNA translation of cellular genes, and that only the first 14 nt in the 5′ end of sgRNA is essential for this process. CRISPR-Cas9 can suppress the protein expression of an unintended target gene without affecting its DNA sequence and causes unexpected phenotypic changes. Using the designed RNA aptamer-ligand complexes which physically obstruct translation machinery, we indicate that roadblock mechanism is responsible for this phenomenon. Our work suggests that studies on Cas9 should avoid the potential off-target effects by detecting the alteration of genes at both the DNA and protein levels.

Highlights

The bacterial type II CRISPR-Cas[9] system is emerging as a versatile tool for basic biology and engineering biology[1,2,3,4,5]
We investigated the effect of single guide RNA (sgRNA)-Cas[9] complex on mRNA translation of cellular genes in human cells and our data revealed that this protein has an additional and yet un-described activity for repression of cellular mRNA translation
We provided direct evidences to test the hypothesis that Cas[9] from S. pyogenes can repress gene expression through inhibition of translation when targeted to RNA sequences that do not have adjacent protospacer adjacent motif (PAM) sequences in the DNA encoding them

Summary

Introduction

The bacterial type II CRISPR-Cas[9] system is emerging as a versatile tool for basic biology and engineering biology[1,2,3,4,5]. Small interfering RNAs , such as siRNAs, shRNAs and microRNAs, mediate gene silencing by directing the RNA-induced silencing complex (RISC) to bind to and degrade the mRNA Based on these observations that mRNA target can be bound by a separate sequence that is complementary to mRNA, and that the sgRNA has an antisense element, we wondered whether the sgRNA-Cas[9] complex can affect the mRNA stability or translation. The translation of HCV RNA is mediated by internal ribosome entry site (IRES), whereas translation of cellular mRNAs is triggered by the 5′-cap structure containing multiple initiation factors It is still unclear whether sgRNA-Cas[9] complex can affect mRNA translation of cellular genes in the absence of PAM-carrying DNA. Reported data were shown as mean ±SD from three biological replicates. *P < 0.05 compared to nonspecific sgRNA control by paired, one-sided t-test

Methods

Results

Conclusion