Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure

Tomoko Nakanishi; Aya Maekawa; Hirotaka Tabata; Takashi Yoshioka; Zheng Pei; Kumiko Sato; Mai Mori; Masaya Kato; Izumu Saito

doi:10.1002/jgm.3115

Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure

J Gene Med. 2019 Nov;21(11):e3115. doi: 10.1002/jgm.3115. Epub 2019 Oct 29.

Authors

Tomoko Nakanishi^{1

2}, Aya Maekawa^{1

2}, Hirotaka Tabata^{1

2

3}, Takashi Yoshioka^{2

4}, Zheng Pei², Kumiko Sato², Mai Mori^{1

2}, Masaya Kato^{1

5}, Izumu Saito^{1

2}

Affiliations

¹ Laboratory of Virology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Foundation, Tokyo, Japan.
² Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
³ Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, Toyama, Japan.
⁴ Department of Urology, Graduate School of Medicine, Okayama University, Okayama, Japan.
⁵ PharmaBio Corporation Co. Ltd., Kawasaki plant, Kawasaki, Japan.

Abstract

Background: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination.

Methods: Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection.

Results: We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites.

Conclusions: We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.

Keywords: cosmid; double nicking; lambda packaging; multiplex guide RNA; non-viral vector; nucleic acid therapy.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacteriophage lambda / genetics
CRISPR-Cas Systems*
Cosmids / genetics*
Gene Amplification*
Gene Editing*
Gene Expression*
Gene Order
Gene Targeting
Hepatitis B virus / genetics
Humans
RNA, Guide, CRISPR-Cas Systems*
Sequence Deletion
Transfection

Substances

RNA, Guide, CRISPR-Cas Systems