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Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

Zexu Li, Zihan Li, Xiaolong Cheng, Xiaofeng Wang, Shixin Ma, Shengnan Wang, Zhiyan Lu, Han Zhang, Wenchang Zhao, Zhisong Chen, Yingjia Yao, Lumen Chao, Wei Li, View ORCID ProfileTeng Fei
doi: https://doi.org/10.1101/2022.05.14.491940
Zexu Li
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Zihan Li
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Xiaolong Cheng
3Center for Genetic Medicine Research, Children’s National Hospital, 111 Michigan Ave NW, Washington, DC, 20010, USA
4Department of Genomics and Precision Medicine, George Washington University, 111 Michigan Ave NW, Washington, DC, 20010, USA
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Xiaofeng Wang
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Shixin Ma
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Shengnan Wang
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Zhiyan Lu
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Han Zhang
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Wenchang Zhao
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Zhisong Chen
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Yingjia Yao
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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Lumen Chao
3Center for Genetic Medicine Research, Children’s National Hospital, 111 Michigan Ave NW, Washington, DC, 20010, USA
4Department of Genomics and Precision Medicine, George Washington University, 111 Michigan Ave NW, Washington, DC, 20010, USA
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Wei Li
3Center for Genetic Medicine Research, Children’s National Hospital, 111 Michigan Ave NW, Washington, DC, 20010, USA
4Department of Genomics and Precision Medicine, George Washington University, 111 Michigan Ave NW, Washington, DC, 20010, USA
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  • For correspondence: wli2@childrensnational.org feiteng@mail.neu.edu.cn
Teng Fei
1College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China
2Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University), Ministry of Education, Shenyang 110819, People’s Republic of China
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  • ORCID record for Teng Fei
  • For correspondence: wli2@childrensnational.org feiteng@mail.neu.edu.cn
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SUMMARY

CRISPR-Cas13 systems have been adapted as versatile toolkits for RNA-related applications. Here we systematically evaluate the performance of several popular Cas13 family effectors (Cas13a, Cas13b and Cas13d) under lentiviral vectors and reveal surprisingly differential defects and characteristics of these systems. Using RNA immunoprecipitation sequencing, transcriptome profiling, biochemistry analysis, high-throughput CRISPR-Cas13 screening and machine learning approaches, we determine that each Cas13 system has its intrinsic RNA targets in mammalian cells. Viral process-related host genes can be targeted by Cas13 and affect production of fertile lentiviral particles, thereby restricting the utility of lentiviral Cas13 systems. Multiple RNase activities of Cas13 are involved in endogenous RNA targeting. Unlike target-induced nonspecific collateral effect, intrinsic RNA cleavage can be specific, target-independent and dynamically tuned by varied states of Cas13 nucleases. Our work provides guidance on appropriate use of lentiviral Cas13 systems and further raises cautions about intrinsic RNA targeting during Cas13-based basic and therapeutic applications.

Competing Interest Statement

W.L. is a paid consultant to Tavros Therapeutics, Inc. Others declared no competing interests.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 14, 2022.
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Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems
Zexu Li, Zihan Li, Xiaolong Cheng, Xiaofeng Wang, Shixin Ma, Shengnan Wang, Zhiyan Lu, Han Zhang, Wenchang Zhao, Zhisong Chen, Yingjia Yao, Lumen Chao, Wei Li, Teng Fei
bioRxiv 2022.05.14.491940; doi: https://doi.org/10.1101/2022.05.14.491940
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Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems
Zexu Li, Zihan Li, Xiaolong Cheng, Xiaofeng Wang, Shixin Ma, Shengnan Wang, Zhiyan Lu, Han Zhang, Wenchang Zhao, Zhisong Chen, Yingjia Yao, Lumen Chao, Wei Li, Teng Fei
bioRxiv 2022.05.14.491940; doi: https://doi.org/10.1101/2022.05.14.491940

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