Efficient modification and preparation of circular DNA for expression in cell culture
Abstract DNA plasmids are an essential tool for delivery and expression of RNAs and proteins in cell culture experiments. The preparation of plasmids typically involves a laborious process of bacterial cloning, validation, and purification. While the expression plasmids can be designed and ordered f...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-12-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-022-04363-z |
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| author | Roman Teo Oliynyk George M. Church |
| author_facet | Roman Teo Oliynyk George M. Church |
| author_sort | Roman Teo Oliynyk |
| collection | DOAJ |
| description | Abstract DNA plasmids are an essential tool for delivery and expression of RNAs and proteins in cell culture experiments. The preparation of plasmids typically involves a laborious process of bacterial cloning, validation, and purification. While the expression plasmids can be designed and ordered from the contract manufacturers, the cost may be prohibitive when a large number of plasmids is required. We have developed an efficient fully synthetic method and protocol that enables the production of circularized DNA containing expression elements ready for transfection in as little as 3 hours, thereby eliminating the bacterial cloning steps. The protocol describes how to take a linear double-stranded DNA fragment and efficiently circularize and purify this DNA fragment with minimal hands-on time. As proof of the principle, we applied Circular Vector expressing engineered prime editing guide RNA (epegRNA) in cell culture, and demonstrated matching and even exceeding performance of this method as compared to guides expressed by plasmids. The method’s speed of preparation, low cost, and ease of use will make it a useful tool in applications requiring the expression of short RNAs and proteins. |
| format | Article |
| id | doaj-art-8adc21a3b23c4b8b8a769ba5ff18d8d7 |
| institution | OA Journals |
| issn | 2399-3642 |
| language | English |
| publishDate | 2022-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-8adc21a3b23c4b8b8a769ba5ff18d8d72025-08-20T02:15:00ZengNature PortfolioCommunications Biology2399-36422022-12-015111010.1038/s42003-022-04363-zEfficient modification and preparation of circular DNA for expression in cell cultureRoman Teo Oliynyk0George M. Church1Department of Genetics, Harvard Medical SchoolDepartment of Genetics, Harvard Medical SchoolAbstract DNA plasmids are an essential tool for delivery and expression of RNAs and proteins in cell culture experiments. The preparation of plasmids typically involves a laborious process of bacterial cloning, validation, and purification. While the expression plasmids can be designed and ordered from the contract manufacturers, the cost may be prohibitive when a large number of plasmids is required. We have developed an efficient fully synthetic method and protocol that enables the production of circularized DNA containing expression elements ready for transfection in as little as 3 hours, thereby eliminating the bacterial cloning steps. The protocol describes how to take a linear double-stranded DNA fragment and efficiently circularize and purify this DNA fragment with minimal hands-on time. As proof of the principle, we applied Circular Vector expressing engineered prime editing guide RNA (epegRNA) in cell culture, and demonstrated matching and even exceeding performance of this method as compared to guides expressed by plasmids. The method’s speed of preparation, low cost, and ease of use will make it a useful tool in applications requiring the expression of short RNAs and proteins.https://doi.org/10.1038/s42003-022-04363-z |
| spellingShingle | Roman Teo Oliynyk George M. Church Efficient modification and preparation of circular DNA for expression in cell culture Communications Biology |
| title | Efficient modification and preparation of circular DNA for expression in cell culture |
| title_full | Efficient modification and preparation of circular DNA for expression in cell culture |
| title_fullStr | Efficient modification and preparation of circular DNA for expression in cell culture |
| title_full_unstemmed | Efficient modification and preparation of circular DNA for expression in cell culture |
| title_short | Efficient modification and preparation of circular DNA for expression in cell culture |
| title_sort | efficient modification and preparation of circular dna for expression in cell culture |
| url | https://doi.org/10.1038/s42003-022-04363-z |
| work_keys_str_mv | AT romanteooliynyk efficientmodificationandpreparationofcirculardnaforexpressionincellculture AT georgemchurch efficientmodificationandpreparationofcirculardnaforexpressionincellculture |