Introducing an All-in-One CRISPR/Cas9 Vector System for Multiplex Genome Engineering

Posted by Kendall Morgan on Nov 12, 2014 10:54:00 AM

CRISPR/Cas9 Multiplex Genome Engineering SystemA newly established all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available thanks to researchers at Japan’s Hiroshima University who described their new tool in Scientific Reports in June.

“The multiplexity is one of the most advantageous properties of CRISPR/Cas9 compared to ZFNs and TALENs,” said Tetsushi Sakuma of Hiroshima University. “However, there had been no systematically established way of making an all-in-one vector for multiplex genome engineering.”

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Now, they’ve filled that gap. As Sakuma and colleague Takashi Yamamoto describe in their recent study, the construction system offers ease and efficiency for all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs). Sakuma said that the new system could also be expanded to include even more loci.

Multiplex Genome Engineering Demonstrated in Human Cells

They used the Golden Gate cloning method to ligate expression cassettes of the gRNAs into a single vector in tandem. They went on to show that their CRISPR/Cas9-nuclease and CRISPR/Cas9-nickase vectors work successfully to target multiple sites in cultured human cells.

While it has not yet been demonstrated, they say the vectors are also likely to work in animal embryos based on earlier evidence showing that a CRISPR/Cas9 plasmid could be injected directly into mice for genome editing. They add that it is also possible that their system may be applied to efficient viral vector-mediated multiplex genome engineering.

The system now provides an efficient targeting strategy for multiplex editing of genomes. It will also allow simultaneous activation and/or repression of multiple genes, and more. The researchers now plan to adapt the new tool for use with a broader array of Cas9s, which they will also make available when they are validated and ready.

“Currently, the kit only includes two versions of Cas9, nuclease and nickase, but we are now adopting dCas9, dCas9-VP64, and dCas9-KRAB in our system,” Sakuma said. “These are sure to be powerful tools to modify the expression of multiple genes at the same time.”

For more details on how to apply the new system, see Yamamoto and Sakuma’s protocol.


Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T. Sci Rep. 2014 Jun 23;4:5400. doi: 10.1038/srep05400. PMID: 24954249

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