Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays

Morito Kurata, Natalie K. Wolf, Walker S. Lahr, Madison T. Weg, Mitchell G. Kluesner, Samantha Lee, Kai Hui, Masano Shiraiwa, Beau R Webber, Branden S Moriarity

Research output: Contribution to journalArticlepeer-review

43 Scopus citations


The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limited due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

Original languageEnglish (US)
Article numbere0198714
JournalPloS one
Issue number9
StatePublished - Sep 2018

Bibliographical note

Funding Information:
This work was supported by the Sobiech Osteosarcoma Fund Award; the Children’s Cancer Research Fund; National Cancer Institute grant R03 1R03CA201502-01.

Publisher Copyright:
© 2018 Kurata et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


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