Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme
Abstract The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR–Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For su...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-09-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-19485-x |
| _version_ | 1811273535834816512 |
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| author | Serene Rose David Sumanth Kumar Maheshwaram Divya Shet Mahesh B. Lakshminarayana Gautam V. Soni |
| author_facet | Serene Rose David Sumanth Kumar Maheshwaram Divya Shet Mahesh B. Lakshminarayana Gautam V. Soni |
| author_sort | Serene Rose David |
| collection | DOAJ |
| description | Abstract The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR–Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min−1 at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9–DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR–Cas9 technology. |
| first_indexed | 2024-04-12T23:01:01Z |
| format | Article |
| id | doaj.art-55cb3d192c104598af89355a07c8f5db |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-12T23:01:01Z |
| publishDate | 2022-09-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-55cb3d192c104598af89355a07c8f5db2022-12-22T03:13:03ZengNature PortfolioScientific Reports2045-23222022-09-0112111010.1038/s41598-022-19485-xTemperature dependent in vitro binding and release of target DNA by Cas9 enzymeSerene Rose David0Sumanth Kumar Maheshwaram1Divya Shet2Mahesh B. Lakshminarayana3Gautam V. Soni4Raman Research InstituteRaman Research InstituteRaman Research InstituteRaman Research InstituteRaman Research InstituteAbstract The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR–Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min−1 at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9–DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR–Cas9 technology.https://doi.org/10.1038/s41598-022-19485-x |
| spellingShingle | Serene Rose David Sumanth Kumar Maheshwaram Divya Shet Mahesh B. Lakshminarayana Gautam V. Soni Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme Scientific Reports |
| title | Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme |
| title_full | Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme |
| title_fullStr | Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme |
| title_full_unstemmed | Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme |
| title_short | Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme |
| title_sort | temperature dependent in vitro binding and release of target dna by cas9 enzyme |
| url | https://doi.org/10.1038/s41598-022-19485-x |
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