Crispr Cas9

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Strategies for controlling CRISPR/Cas9 off-target effects and biological variations in mammalian genome editing experiments.

Highlights

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CRISPR/Cas9 off-target effects (OTEs) can confound interpretation of results.
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Considerations on picking proper host cells and genes for CRISPRediting discussed.
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Mitigation of OTEs via gRNA design and controlled exposure of Cas9/gRNAs discussed.
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Approaches on controlling CRISPR OTEs and biological variations discussed.
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Strategies discussed here applicable to all genome editingexperiments.

Abstract

The CRISPR/Cas9 system has enabled efficient modification of genes in a variety of cellular systems for studying phenotypic effects of genetic perturbations. However, with this technology comes the inherent risk of generating off-target effects (OTEs) in addition to the desired modifications. As such, it can be difficult to conclusively determine that the observed phenotypic changes are in fact due to the intended modification of the target gene and not from random mutations elsewhere in the genome. In addition, biological variations observed within cultured cells or laboratory animals can also confound results and need to be addressed. In this article, we review potential sources of experimental and biological variation as well as propose experimental options to minimize and control OTEs and other variations in CRISPR genome editing experiments for exploratory research applications. Confirmation of on-target KO effect by orthogonal approaches is also discussed.

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