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

Sci Rep. 2018 Jan 8;8(1):93. doi: 10.1038/s41598-017-18551-z.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Cell Death / genetics
  • Cell Line, Tumor
  • Disease Models, Animal
  • Gene Knockdown Techniques
  • Glioma / genetics
  • Glioma / metabolism
  • Glioma / pathology
  • Heterografts
  • Humans
  • Mice
  • RNA Interference
  • RNA, Small Interfering / genetics*
  • Semaphorins / genetics
  • Semaphorins / metabolism


  • RNA, Small Interfering
  • Semaphorins
  • semaphorin 4B, human