Combined shRNA over CRISPR/cas9 as a methodology to detect off-target effects and a potential compensatory mechanism

Liat Peretz, Elazar Besser, Renana Hajbi, Natania Casden, Dan Ziv, Nechama Kronenberg, Liat Ben Gigi, Sahar Sweetat, Saleh Khawaled, Rami Aqeilan, Oded Behar*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

Inhibition of genes is a powerful approach to study their function. While RNA interference is a widely used method to achieve this goal, mounting evidence indicates that such an approach is prone to off-target effects. An alternative approach to gene function inhibition is genetic mutation, such as the CRISPR/cas9 method. A recent report, however, demonstrated that genetic mutation and inhibition of gene expression do not always give corresponding results. This can be explained by off-target effects, but it was recently shown, at least in one case, that these differences are the result of a compensatory mechanism induced only by genetic mutation. We present here a combination of RNA inhibition and CRISPR/cas9 methods to identify possible off targets as well as potential compensatory effects. This approach is demonstrated by testing a possible role for Sema4B in glioma biology, in which our results implicate Sema4B as having a critical function. In stark contrast, by using shRNA over CRISPR/cas9 combined methodology, we clearly demonstrate that the Sema4B targeted shRNA effects on cell proliferation is the result of off-target effects. Nevertheless, it also revealed that certain splice variants of Sema4B are important for the ability of glioma cells to grow as individual clones.

Original languageAmerican English
Article number93
JournalScientific Reports
Volume8
Issue number1
DOIs
StatePublished - 1 Dec 2018

Bibliographical note

Funding Information:
We are grateful to Dr. Norman Grover (Department of Experimental Medicine, The Hebrew University) for helpful advice regarding the statistical analyses. We would also like to thank Dr. Acker Till (Institute of Neuropathology Medicine, University Giessen, Germany) for providing the G55TL primary glioblastoma cells. This work was supported by a grant from the Israel Science Foundation (Grant No. 947/14) and from the Israel Cancer Research Fund (Grant No. 01948).

Publisher Copyright:
© 2017 The Author(s).

Fingerprint

Dive into the research topics of 'Combined shRNA over CRISPR/cas9 as a methodology to detect off-target effects and a potential compensatory mechanism'. Together they form a unique fingerprint.

Cite this