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Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing

Bin Li, Chunxi Zeng, Wenqing Li, Xinfu Zhang, Xiao Luo, Weiyu Zhao, Chengxiang Zhang, Yizhou Dong
doi: https://doi.org/10.1101/253757
Bin Li
1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
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Chunxi Zeng
1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
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Wenqing Li
1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
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Xinfu Zhang
1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
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Xiao Luo
1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
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Weiyu Zhao
1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
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Chengxiang Zhang
1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
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Yizhou Dong
1Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
2Department of Biomedical Engineering, The Ohio State University, Columbus, OH 43210, USA.
3The Center for Clinical and Translational Science, The Ohio State University, Columbus, OH 43210, USA.
4James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
5Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA.
6Department of Radiation Oncology, The Ohio State University, Columbus, OH 43210, USA.
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Abstract

CRISPR-Cpf1, a microbial adaptive immune system discovered from Prevotella and Francisella 1, employs a single-stranded CRISPR RNA (crRNA) to induce double stranded DNA breaks1. To modulate genome editing activity of Cpf1 in human cells, we designed a series of crRNA variants including DNA-crRNA and RNA-crRNA duplexes, and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1 mediated gene editing. More importantly, without prehybridization, this PS-modified DNA was able to regulate Cpf1 activity in a time-and dose-dependent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder the binding between Cpf1-crRNA complex and target DNA substrate. Consequently, phosphorothioate-modified DNA oligonucleotides provide a tunable platform to inactivate Cpf1 mediated genome editing.

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Posted February 06, 2018.
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Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing
Bin Li, Chunxi Zeng, Wenqing Li, Xinfu Zhang, Xiao Luo, Weiyu Zhao, Chengxiang Zhang, Yizhou Dong
bioRxiv 253757; doi: https://doi.org/10.1101/253757
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Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing
Bin Li, Chunxi Zeng, Wenqing Li, Xinfu Zhang, Xiao Luo, Weiyu Zhao, Chengxiang Zhang, Yizhou Dong
bioRxiv 253757; doi: https://doi.org/10.1101/253757

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