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

LION: a simple and rapid method to achieve CRISPR gene editing.
Cell Mol Life Sci. 2019 Mar 18;:
Authors: Xiang X, Luo L, Nodzyński M, Li C, Han P, Dou H, Petersen TS, Liang X, Pan X, Qu K, Yang L, Dang Y, Liu X, Bolund L, Zhang X, Tong G, Xing Y, Luo Y, Lin L
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.
PMID...

Source: Cellular and Molecular Life Sciences : CMLS - March 17, 2019 Category: Cytology Authors: Xiang X, Luo L, Nodzyński M, Li C, Han P, Dou H, Petersen TS, Liang X, Pan X, Qu K, Yang L, Dang Y, Liu X, Bolund L, Zhang X, Tong G, Xing Y, Luo Y, Lin L Tags: Cell Mol Life Sci Source Type: research