Abstract
The engineered CRISPR/Cas9 technology has developed as the most efficient and broadly used genome editing tool. However, simultaneously targeting multiple genes (or genomic loci) in the same individual cells using CRISPR/Cas9 remain one technical challenge. In this article, we have developed a Golden Gate Assembly method for the generation of CRISPR gRNA expression arrays, thus enabling simultaneous gene targeting. Using this method, the generation of CRISPR gRNA expression array can be accomplished in 2 weeks, and contains up to 30 gRNA expression cassettes. We demonstrated in the study that simultaneously targeting 10 genomic loci or simultaneously inhibition of multiple endogenous genes could be achieved using the multiplexed gRNA expression array vector in human cells. The complete set of plasmids is available through the non-profit plasmid repository Addgene.
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Notes
All gRNA expression cassettes in our method are driven by the U6 promoter.
It is very important to treat the reaction with plasmid safe DNA nuclease when assembling more than five gRNA expression cassettes. This step is to avoid the recombination of the linearized DNA fragments following transformation.
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Acknowledgments
JVN and ALN were supported by grants from The Lundbeck Foundation, Krista og Viggo Petersens Fond, Fabrikant Einar Willumsens Mindelegat, and Fonden til Lægevidenskabens Fremme. LL and LB were supported by the DREAM project from Lundbeck Foundation. YL was supported by grants from Danish Research Council for Independent Research, the Sapere Aude Young Research Talent prize to YL, the Lundbeck Foundation and the Innovation Fund Denmark (BrainStem).
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Vad-Nielsen, J., Lin, L., Bolund, L. et al. Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes. Cell. Mol. Life Sci. 73, 4315–4325 (2016). https://doi.org/10.1007/s00018-016-2271-5
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DOI: https://doi.org/10.1007/s00018-016-2271-5