High-throughput screen reveals sRNAs regulating crRNA biogenesis by targeting CRISPR leader to repress Rho termination

Ping Lin; Qinqin Pu; Qun Wu; Chuanmin Zhou; Biao Wang; Jacob Schettler; Zhihan Wang; Shugang Qin; Pan Gao; Rongpeng Li; Guoping Li; Zhenyu Cheng; Lefu Lan; Jianxin Jiang; Min Wu

doi:10.1038/s41467-019-11695-8

High-throughput screen reveals sRNAs regulating crRNA biogenesis by targeting CRISPR leader to repress Rho termination

Ping Lin, Qinqin Pu, Qun Wu, Chuanmin Zhou, Biao Wang, Jacob Schettler, Zhihan Wang, Shugang Qin, Pan Gao, Rongpeng Li, Guoping Li, Zhenyu Cheng, Lefu Lan, Jianxin Jiang, Min Wu

Research output: Contribution to journal › Article › peer-review

29 Citations (Scopus)

Abstract

Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.

Original language	English
Article number	3728
Journal	Nature Communications
Volume	10
Issue number	1
DOIs	https://doi.org/10.1038/s41467-019-11695-8
Publication status	Published - Dec 1 2019

Bibliographical note

Funding Information:
We thank Dr. Stephen Lory of Harvard Medical School to give pKH6, pKH13, or lacZ vectors, Dr. Evgeny Nudler of New York University School of Medicine for providing Rho and NusG proteins, Dr. Elisabeth Sonnleitner of University of Vienna for providing pJT-phrS, pJT-phrSΔcreg, pJT-phrScreg, and pJT-phrScmut, Dr. Alan R. Davidson of University of Toronto for providing Pseudomonas aeruginosa SMC4386, Dr. Shiyun Chen of Wuhan institute of Virology (Chinese Academy of Science) for providing information of sRNA library information, and Dr. Sergei Nechaev and Dr. Catherine Brissette of University of North Dakota for critical reading. This work was supported by National Institutes of Health Grants AI101973-01, R01AI109317-01A1, and R01AI0138203-01 to M.W., UND Post-Doc Pilot Grant; this work was also supported by the Key Program of National Nature Science Foundation of China (81530063) to Jianxin Jiang. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Publisher Copyright:
© 2019, The Author(s).

ASJC Scopus Subject Areas

General Chemistry
General Biochemistry,Genetics and Molecular Biology
General Physics and Astronomy

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title = "High-throughput screen reveals sRNAs regulating crRNA biogenesis by targeting CRISPR leader to repress Rho termination",

abstract = "Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.",

author = "Ping Lin and Qinqin Pu and Qun Wu and Chuanmin Zhou and Biao Wang and Jacob Schettler and Zhihan Wang and Shugang Qin and Pan Gao and Rongpeng Li and Guoping Li and Zhenyu Cheng and Lefu Lan and Jianxin Jiang and Min Wu",

note = "Funding Information: We thank Dr. Stephen Lory of Harvard Medical School to give pKH6, pKH13, or lacZ vectors, Dr. Evgeny Nudler of New York University School of Medicine for providing Rho and NusG proteins, Dr. Elisabeth Sonnleitner of University of Vienna for providing pJT-phrS, pJT-phrSΔcreg, pJT-phrScreg, and pJT-phrScmut, Dr. Alan R. Davidson of University of Toronto for providing Pseudomonas aeruginosa SMC4386, Dr. Shiyun Chen of Wuhan institute of Virology (Chinese Academy of Science) for providing information of sRNA library information, and Dr. Sergei Nechaev and Dr. Catherine Brissette of University of North Dakota for critical reading. This work was supported by National Institutes of Health Grants AI101973-01, R01AI109317-01A1, and R01AI0138203-01 to M.W., UND Post-Doc Pilot Grant; this work was also supported by the Key Program of National Nature Science Foundation of China (81530063) to Jianxin Jiang. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: {\textcopyright} 2019, The Author(s).",

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doi = "10.1038/s41467-019-11695-8",

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AU - Lin, Ping

AU - Pu, Qinqin

AU - Wu, Qun

AU - Zhou, Chuanmin

AU - Wang, Biao

AU - Schettler, Jacob

AU - Wang, Zhihan

AU - Qin, Shugang

AU - Gao, Pan

AU - Li, Rongpeng

AU - Li, Guoping

AU - Cheng, Zhenyu

AU - Lan, Lefu

AU - Jiang, Jianxin

AU - Wu, Min

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PY - 2019/12/1

Y1 - 2019/12/1

N2 - Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.

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High-throughput screen reveals sRNAs regulating crRNA biogenesis by targeting CRISPR leader to repress Rho termination

Abstract

Bibliographical note

ASJC Scopus Subject Areas

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