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Escape phenomenon of CRISPR/Cas13a system during RNA editing in Escherichia coli
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Yue ZHANG1, 2, 3, Jie XU2, 3, Hengyu WANG2, 3, Yong SHENG2, 3, Yixin OU2, 3, Bin WANG1, Bei ZHANG1, Qianjin KANG2, 3, *, Li ZHANG1, *
Acta Microbiologica Sinica | 2024, 64(8) : 2998 - 3013
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Acta Microbiologica Sinica | 2024, 64(8): 2998-3013
• Research Articles •
Escape phenomenon of CRISPR/Cas13a system during RNA editing in Escherichia coli
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Yue ZHANG1, 2, 3, Jie XU2, 3, Hengyu WANG2, 3, Yong SHENG2, 3, Yixin OU2, 3, Bin WANG1, Bei ZHANG1, Qianjin KANG2, 3, *, Li ZHANG1, *
Affiliations
  • 1 School of Basic Medicine, Qingdao Medical College, Qingdao University, Qingdao 266071, Shandong, China
  • 2 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
  • 3 Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University, Shanghai 200240, China
Published: 2024-04-08 doi: 10.13343/j.cnki.wsxb.20240081
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[Objective] The plasmid interference system of CRISPR/LshCas13a was constructed in Escherichia coli MG1655-ΔrecA and Escherichia coli DH10B to analyze the escape phenomenon in RNA editing experiments by targeting the non-essential gene lacZ and the essential gene polA. [Methods] An inducible CRISPR/LshCas13a RNA editing system- associated plasmid was designed with LshCas13a from Leptotrichia shahii. MG1655-ΔrecA and DH10B were selected as the research objects. The Crisporo algorithm was employed to design the CRISPR RNA (crRNA) sequences targeting lacZ and polA, and the LshCas13a plasmid interference experiment was carried out to study the escape phenomena targeting lacZ and polA. The escape phenomenon of the LshCas13a system was evaluated based on the number and sequences of escaped colonies. PCR and Sanger sequencing were conducted to explore the escape events of the LshCas13a system. The escaped colonies carrying the LshCas13a system disrupted by the insertion sequence (IS) were selected, and OD600 was measured to evaluate the growth recovery of the strains. [Results] The LshCas13a system was used to target lacZ and polA in MG1655-ΔrecA and DH10B. MG1655-ΔrecA escaped through point mutation of LshCas13a and IS-mediated transposition when lacZ was targeted. When polA was targeted, MG1655-ΔrecA and DH10B escaped by point mutation of LshCas13a, IS-mediated transposition, and mutation of the direct repeat (DR) sequence of crRNA. The mutation of LshCas13a promoted the recovery of strain growth. [Conclusion] The LshCas13a plasmid interference system successfully revealed the diversified escape phenomena during the RNA editing of E. coli, including IS-mediated the transposition of LshCas13a, point mutation of LshCas13a, and DR sequence mutation or recombination of crRNA. The results laid a foundation for optimization of the CRISPR/LshCas13a gene editing system.

Escherichia coli  /  CRISPR/LshCas13a  /  plasmid interference system  /  escape phenomenon  /  insertion sequence
Yue ZHANG, Jie XU, Hengyu WANG, Yong SHENG, Yixin OU, Bin WANG, Bei ZHANG, Qianjin KANG, Li ZHANG. Escape phenomenon of CRISPR/Cas13a system during RNA editing in Escherichia coli[J]. Acta Microbiologica Sinica, 2024 , 64 (8) : 2998 -3013 . DOI: 10.13343/j.cnki.wsxb.20240081
Funding
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  • National Key Research and Development Program of China(2021YFC2100600)
  • Agricultural Science and Technology Innovation Program of Shanghai(202302080012F04596)
  • "Major Project" of Haihe Laboratory of Synthetic Biology(22HHSWSS00001)
Appendix
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Year 2024 volume 64 Issue 8
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Article Info
doi: 10.13343/j.cnki.wsxb.20240081
  • Receive Date：2024-01-31
  • Online Date：2026-03-19
  • Published：2024-04-08
Article Data
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History
  • Received：2024-01-31
  • Accepted：2024-04-02
Funding
National Key Research and Development Program of China(2021YFC2100600)
Agricultural Science and Technology Innovation Program of Shanghai(202302080012F04596)
"Major Project" of Haihe Laboratory of Synthetic Biology(22HHSWSS00001)
Affiliations
    1 School of Basic Medicine, Qingdao Medical College, Qingdao University, Qingdao 266071, Shandong, China
    2 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
    3 Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University, Shanghai 200240, China

Corresponding:

*KANG Qianjin, E-mail:
ZHANG Li, E-mail:
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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