Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit
Abstract Background Plasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or app...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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BMC
2017-09-01
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| Series: | Journal of Biological Engineering |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13036-017-0072-5 |
| _version_ | 1818411375902326784 |
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| author | Qiang Tang Chunbo Lou Shuang-Jiang Liu |
| author_facet | Qiang Tang Chunbo Lou Shuang-Jiang Liu |
| author_sort | Qiang Tang |
| collection | DOAJ |
| description | Abstract Background Plasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or application. Conventional strategies including exposure to physical and chemical treatments, or exploiting temperature-sensitive replication origins have several drawbacks (e.g., they are limited for efficiency and are time-consuming). Therefore, the demand is apparent for easy and rapid elimination of the tool plasmids from their bacterial hosts after genetic manipulation. Results To bridge this gap, we designed a novel EXIT circuit with the homing endonuclease, which can be exploited for rapid and efficient elimination of various plasmids with diverse replication origins. As a proof of concept, we validated the EXIT circuit in Escherichia coli by harnessing homing endonuclease I-SceI and its cleavage site. When integrated into multiple plasmids with different origins, the EXIT circuit allowed them to be eliminated from the host cells, simultaneously. By combining the widely used plasmid-borne CRISPR-Cas9 system and the EXIT circuit, we constructed an easy-to-use CRISPR-Cas9 system that eliminated the Cas9- and the single-guide RNA (sgRNA)-encoding plasmids in one-step. Within 3 days, we successfully constructed an atrazine-degrading E. coli strain, thus further demonstrating the advantage of this new CRISPR-Cas9 system for bacterial genome editing. Conclusions Our novel EXIT circuit, which exploits the homing endonuclease I-SceI, enables plasmid(s) with different replication origins to be eliminated from their host cells rapidly and efficiently. We also developed an easy-to-use CRISPR-Cas9 system with the EXIT circuit, and this new system can be widely applied to bacterial genome editing. |
| first_indexed | 2024-12-14T10:30:25Z |
| format | Article |
| id | doaj.art-bdbc006d5e2147309c26ce4ae1f8a461 |
| institution | Directory Open Access Journal |
| issn | 1754-1611 |
| language | English |
| last_indexed | 2024-12-14T10:30:25Z |
| publishDate | 2017-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Biological Engineering |
| spelling | doaj.art-bdbc006d5e2147309c26ce4ae1f8a4612022-12-21T23:06:10ZengBMCJournal of Biological Engineering1754-16112017-09-0111111110.1186/s13036-017-0072-5Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuitQiang Tang0Chunbo Lou1Shuang-Jiang Liu2State Key Laboratory of Microbial Resources and Environmental Microbiology Research Center, Chinese Academy of SciencesCAS Key Laboratory for Microbial Physiology and Metabolic Engineering, Chinese Academy of SciencesState Key Laboratory of Microbial Resources and Environmental Microbiology Research Center, Chinese Academy of SciencesAbstract Background Plasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or application. Conventional strategies including exposure to physical and chemical treatments, or exploiting temperature-sensitive replication origins have several drawbacks (e.g., they are limited for efficiency and are time-consuming). Therefore, the demand is apparent for easy and rapid elimination of the tool plasmids from their bacterial hosts after genetic manipulation. Results To bridge this gap, we designed a novel EXIT circuit with the homing endonuclease, which can be exploited for rapid and efficient elimination of various plasmids with diverse replication origins. As a proof of concept, we validated the EXIT circuit in Escherichia coli by harnessing homing endonuclease I-SceI and its cleavage site. When integrated into multiple plasmids with different origins, the EXIT circuit allowed them to be eliminated from the host cells, simultaneously. By combining the widely used plasmid-borne CRISPR-Cas9 system and the EXIT circuit, we constructed an easy-to-use CRISPR-Cas9 system that eliminated the Cas9- and the single-guide RNA (sgRNA)-encoding plasmids in one-step. Within 3 days, we successfully constructed an atrazine-degrading E. coli strain, thus further demonstrating the advantage of this new CRISPR-Cas9 system for bacterial genome editing. Conclusions Our novel EXIT circuit, which exploits the homing endonuclease I-SceI, enables plasmid(s) with different replication origins to be eliminated from their host cells rapidly and efficiently. We also developed an easy-to-use CRISPR-Cas9 system with the EXIT circuit, and this new system can be widely applied to bacterial genome editing.http://link.springer.com/article/10.1186/s13036-017-0072-5EXIT circuitI-SceIPlasmid eliminationOne-stepCRISPR-Cas9Easy-to-use |
| spellingShingle | Qiang Tang Chunbo Lou Shuang-Jiang Liu Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit Journal of Biological Engineering EXIT circuit I-SceI Plasmid elimination One-step CRISPR-Cas9 Easy-to-use |
| title | Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit |
| title_full | Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit |
| title_fullStr | Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit |
| title_full_unstemmed | Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit |
| title_short | Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit |
| title_sort | construction of an easy to use crispr cas9 system by patching a newly designed exit circuit |
| topic | EXIT circuit I-SceI Plasmid elimination One-step CRISPR-Cas9 Easy-to-use |
| url | http://link.springer.com/article/10.1186/s13036-017-0072-5 |
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