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LION: a simple and rapid method to achieve CRISPR gene editing

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Abstract

The RNA-guided CRISPR–Cas9 technology has paved the way for rapid and cost-effective gene editing. However, there is still a great need for effective methods for rapid generation and validation of CRISPR/Cas9 gRNAs. Previously, we have demonstrated that highly efficient generation of multiplexed CRISPR guide RNA (gRNA) expression array can be achieved with Golden Gate Assembly (GGA). Here, we present an optimized and rapid method for generation and validation in less than 1 day of CRISPR gene targeting vectors. The method (LION) is based on ligation of double-stranded gRNA oligos into CRISPR vectors with GGA followed by nucleic acid purification. Using a dual-fluorescent reporter vector (C-Check), T7E1 assay, TIDE assay and a traffic light reporter assay, we proved that the LION-based generation of CRISPR vectors are functionally active, and equivalent to CRISPR plasmids generated by traditional methods. We also tested the activity of LION CRISPR vectors in different human cell types. The LION method presented here advances the rapid functional validation and application of CRISPR system for gene editing and simplified the CRISPR gene-editing procedures.

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Abbreviations

CRISPR:

Clustered regularly interspaced short palindromic repeats

Cas9:

CRISPR-associated protein 9

gRNA:

Guide RNA

DSB:

DNA double-stranded break

TIDE:

Tracking indels by decomposition

C-Check:

A dual-fluorescent surrogate reporter system for checking CRISPR cleavage activity in vivo

GGA:

Golden Gate Assembly

LION:

LIgation of double-stranded gRNA oligos into CRISPR vector by GGA followed by nucleic acid purificatiON

SpCas9-RecA:

Streptococcus pyogenes Cas9 fused to E. coli recombinant RecA protein

TLR:

Traffic light reporter

FCM:

Flow cytometry

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Acknowledgements

We thank Fred Dubee for his generous help with language editing. This project was supported by the Sanming Project of Medicine in Shenzhen (SZSM201612074), BGI-Qingdao, and BGI-Shenzhen. Y.L was partially supported by the Danish Research Council for Independent Research (DFF–1337-00128), the Sapere Aude Young Research Talent Prize (DFF-1335-00763A), and Aarhus University Strategic Grant (AU-iCRISPR). L.L. was supported by the Lundbeck Foundation (R219-2016-1375) and the DFF Sapere Aude Starting Grant (8048-00072A).

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Authors and Affiliations

  1. BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, 518083, China

    Xi Xiang, Conghui Li & Xiuqing Zhang

  2. Lars Bolund Institute of Regenerative Medicine, BGI-Qingdao, BGI-Shenzhen, Qingdao, 266555, China

    Xi Xiang, Conghui Li, Peng Han, Hongwei Dou, Xue Liang, Xiaoguang Pan, Kunli Qu, Ling Yang, Yonghui Dang, Xin Liu, Lars Bolund, Xiuqing Zhang, Yonglun Luo & Lin Lin

  3. Department of Biomedicine, Aarhus University, 8000, Aarhus, Denmark

    Xi Xiang, Trine Skov Petersen, Lars Bolund, Yonglun Luo & Lin Lin

  4. BGI-Shenzhen, Shenzhen, 518083, China

    Xi Xiang, Hongwei Dou, Ling Yang, Xin Liu, Xiuqing Zhang & Yonglun Luo

  5. Department of Liver Disease, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, 518033, China

    Xi Xiang, Lidan Luo, Guangdong Tong & Yufeng Xing

  6. Department of General Biochemistry, Jagiellonian University, ul. Gronostajowa 7, 30-387, Kraków, Poland

    Michał Nodzyński, Lars Bolund & Xiuqing Zhang

  7. China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China

    Xin Liu, Lars Bolund & Yonglun Luo

  8. College of Forensics and Medicine, Xi’an Jiaotong University Health Science Centre, Xi’an, 710049, China

    Yonghui Dang

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  1. Xi Xiang
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Correspondence to Yufeng Xing, Yonglun Luo or Lin Lin.

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Xiang, X., Luo, L., Nodzyński, M. et al. LION: a simple and rapid method to achieve CRISPR gene editing. Cell. Mol. Life Sci. 76, 2633–2645 (2019). https://doi.org/10.1007/s00018-019-03064-x

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  • DOI: https://doi.org/10.1007/s00018-019-03064-x

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