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

Xi Xiang, Lidan Luo, Michał Nodzyński, Conghui Li, Peng Han, Hongwei Dou, Trine Skov Petersen, Xue Liang, Xiaoguang Pan, Kunli Qu, Ling Yang, Yonghui Dang, Xin Liu, Lars Bolund, Xiuqing Zhang, Guangdong Tong, Yufeng Xing, Yonglun Luo, Lin Lin

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

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.

Original languageEnglish
JournalCellular and Molecular Life Sciences
Volume76
Issue13
Pages (from-to)2633-2645
Number of pages13
ISSN1420-682X
DOIs
Publication statusPublished - Jul 2019

Keywords

  • CRISPR
  • CRISPR delivery
  • CRISPR efficiency
  • Cas9
  • Golden Gate Assembly

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