Cao Jian, Wu Lizhen, Zhang Shang-Min, Lu Min, Cheung William K C, Cai Wesley, Gale Molly, Xu Qi, Yan Qin
Department of Pathology, Yale School of Medicine, New Haven, CT, 06520 USA.
Harbin Institute of Technology, Harbin, 150001 China.
Nucleic Acids Res. 2016 Nov 2;44(19):e149. doi: 10.1093/nar/gkw660. Epub 2016 Jul 25.
The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack of easy solutions for multiplex targeting, are still limiting its applications. To overcome these challenges, we first developed a highly efficient doxycycline-inducible Cas9-EGFP vector. This vector allowed us to track the cells for uniform temporal control and efficient gene disruption, even in a polyclonal setting. Furthermore, the inducible CRISPR/Cas9 system dramatically decreased off-target effects with a pulse exposure of the genome to the Cas9/sgRNA complex. To target multiple genes simultaneously, we established simple one-step cloning approaches for expression of multiple sgRNAs with improved vectors. By combining our inducible and multiplex genome editing approaches, we were able to simultaneously delete Lysine Demethylase (KDM) 5A, 5B and 5C efficiently in vitro and in vivo This user friendly and highly efficient toolbox provides a solution for easy genome editing with tight temporal control, minimal off-target effects and multiplex targeting.
CRISPR/Cas9系统是一种强大的基因组编辑工具，已广泛应用于生物医学研究。然而，许多挑战，如脱靶效应和缺乏针对多重靶向的简便解决方案，仍在限制其应用。为了克服这些挑战，我们首先开发了一种高效的强力霉素诱导型Cas9-EGFP载体。该载体使我们能够追踪细胞，以实现统一的时间控制和高效的基因破坏，即使在多克隆环境中也是如此。此外，诱导型CRISPR/Cas9系统通过将基因组短暂暴露于Cas9/sgRNA复合物，显著降低了脱靶效应。为了同时靶向多个基因，我们建立了简单的一步克隆方法，用于用改进的载体表达多个sgRNA。通过结合我们的诱导型和多重基因组编辑方法，我们能够在体外和体内高效地同时删除赖氨酸去甲基化酶（KDM）5A、5B和5C。这个用户友好且高效的工具箱为在严格的时间控制、最小的脱靶效应和多重靶向条件下轻松进行基因组编辑提供了解决方案。
