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

Scientific Reports
Liat PeretzOded Behar

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 ab...Continue Reading

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Citations

Mar 11, 2020·PloS One·Morgane CanonnePatricia Renard
Sep 11, 2019·Current Protocols in Molecular Biology·Christopher J GiulianoJason M Sheltzer
Aug 5, 2019·Cellular and Molecular Life Sciences : CMLS·Blanca Martínez-ArribasDolores González-Pacanowska
Oct 20, 2020·Adipocyte·Markus MandlWerner Zwerschke
May 21, 2021·Briefings in Bioinformatics·Thomas GaudeletJake P Taylor-King

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Methods Mentioned

BETA
xenograft
xenografts
PCR
electrophoresis
ELISA

Software Mentioned

Image Pro Plus

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