Journal
CELLULAR AND MOLECULAR LIFE SCIENCES
Volume 76, Issue 13, Pages 2633-2645Publisher
SPRINGER BASEL AG
DOI: 10.1007/s00018-019-03064-x
Keywords
CRISPR; Cas9; Golden Gate Assembly; CRISPR efficiency; CRISPR delivery
Categories
Funding
- Sanming Project of Medicine in Shenzhen [SZSM201612074]
- BGI-Shenzhen
- Danish Research Council for Independent Research [DFF-1337-00128]
- Sapere Aude Young Research Talent Prize [DFF-1335-00763A]
- Aarhus University Strategic Grant (AU-iCRISPR)
- Lundbeck Foundation [R219-2016-1375]
- DFF Sapere Aude Starting Grant [8048-00072A]
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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 1day 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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