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Journal of Integrative Agriculture  2019, Vol. 18 Issue (12): 2835-2843    DOI: 10.1016/S2095-3119(19)62744-9
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
Truncated gRNA reduces CRISPR/Cas9-mediated off-target rate for MSTN gene knockout in bovines
ZHOU Zheng-wei*, CAO Guo-hua*, LI Zhe*, HAN Xue-jie, LI Chen, LU Zhen-yu, ZHAO Yu-hang, LI Xue-ling 
State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestocks/Research Center for Laboratory Animal Science, Inner Mongolia University, Hohhot 010070, P.R.China
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The CRISPR/Cas9 mediates efficient gene editing but has off-target effects inconducive to animal breeding.  In this study, the efficacy of CRISPR/Cas9 vectors containing different lengths of gRNA in reduction of the off-target phenomenon in the bovine MSTN gene knockout fibroblast cell lines was assessed, providing insight into improved methods for livestock breeding.  A 20-bp gRNA was designed for the second exon of the bovine MSTN gene, and CRISPR/Cas9-B was constructed to guide the Cas9 protein to the AGAACCAGGAGAAGATGGACTGG site.  The alternative CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 vectors were constructed using gRNAs truncated by 1, 2, 3 and 5 bp, respectively.  These vectors were then introduced into bovine fetal fibroblasts by the electroporation method, and single cells were obtained by flow cytometry sorting.  PCR was performed for each off-target site.  All samples were sequenced and analyzed, and finally the efficiency of each vector in target and off-target sites was compared.  The CRISPR/Cas9-B vector successfully knocked out the MSTN gene, but the off-target phenomenon was observed.  The efficiencies of CRISPR/Cas-B, CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 in triggering gene mutations at MSTN targeting sites were 62.16, 17.39, 7.69, 74.29 and 3.85%, respectively; rates of each at the Off-MSTN-1 locus were 52.86, 0, 0, 8.82 and 0%, respectively; all were 0% at the Off-MSTN-2 locus; rates at the Off-MSTN-3 site were 44.87, 51.72, 86.36, 0 and 50%, respectively.  The efficiency of the CRISPR/Cas9-17 plasmid in the MSTN site was higher than that in the CRISPR/Cas9-B plasmid, and the effect at the three off-target sites was significantly lower.  This study demonstrated that the CRISPR/Cas9-17 plasmid constructed by truncating 3 bp gRNA can effectively reduce the off-target effect without reducing the efficiency of bovine MSTN gene targeting.  This finding will provide more effective gene editing strategy for use of CRISPR/Cas9 technology.
Keywords:  CRISPR/Cas9        gRNA        targeting site        off-target rate
Received: 19 October 2018   Accepted:
Fund: This study was supported by the National Transgenic Project of China (2016ZX08010001-002 and 2016ZX08010005-001), the National Natural Science Foundation of China (81471001), and the Inner Mongolia Science and Technology Program, China (201502073).
Corresponding Authors:  Correspondence LI Xue-ling, Tel: +86-471-3679807, E-mail:    
About author:  ZHOU Zheng-wei, E-mail:; * These authors contributed equally to this study.

Cite this article: 

ZHOU Zheng-wei, CAO Guo-hua, LI Zhe, HAN Xue-jie, LI Chen, LU Zhen-yu, ZHAO Yu-hang, LI Xue-ling. 2019. Truncated gRNA reduces CRISPR/Cas9-mediated off-target rate for MSTN gene knockout in bovines. Journal of Integrative Agriculture, 18(12): 2835-2843.

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