CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter
- Yuda Wei1,2,
- Yan Qiu1,2,
- Yanhao Chen1,2,
- Gaigai Liu1,2,
- Yongxian Zhang1,2,
- Luwei Xu3 and
- Qiurong Ding1,2
- 1CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
- 2University of Chinese Academy of Sciences, Shanghai 200031, China
- 3Taizhou Hospital of Traditional Chinese Medicine, Taizhou 225300, China
- Corresponding author: qrding{at}sibs.ac.cn
Abstract
Multiplex genome engineering in vivo with CRISPR/Cas9 shows great promise as a potential therapeutic approach. The ability to incorporate multiple single guide RNA (sgRNA) cassettes together with Cas9 gene expression in one AAV vector could greatly enhance the efficiency. In a recent Method article, Mefferd and coworkers indicated that small tRNA promoters could be used to drive sgRNA expression to facilitate the construction of a more effective AAV vector. In contrast, we found that when targeting endogenous genomic loci, CRISPR/Cas9 with tRNA promoter-driven sgRNA expression showed much reduced genome editing activity, compared with significant cleavage with U6 promoter-driven sgRNA expression. Though the underlying mechanisms are still under investigation, our study suggests that the CRISPR/Cas9 system with tRNA promoter-driven sgRNA expression needs to be reevaluated before it can be used for therapeutic genome editing.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.057596.116.
- Received May 18, 2016.
- Accepted October 10, 2016.
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