Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis
CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites availab...
| Main Authors: | , , |
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| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
| Published: |
2016
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| Online Access: | https://hdl.handle.net/10356/84342 http://hdl.handle.net/10220/41788 |
| _version_ | 1824455972682203136 |
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| author | Lee, Raymond Teck Ho Ng, Ashley Shu Mei Ingham, Philip William |
| author2 | Fujii, Hodaka |
| author_facet | Fujii, Hodaka Lee, Raymond Teck Ho Ng, Ashley Shu Mei Ingham, Philip William |
| author_sort | Lee, Raymond Teck Ho |
| collection | NTU |
| description | CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish. |
| first_indexed | 2025-02-19T03:46:42Z |
| format | Journal Article |
| id | ntu-10356/84342 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2025-02-19T03:46:42Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | ntu-10356/843422020-11-01T05:31:50Z Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis Lee, Raymond Teck Ho Ng, Ashley Shu Mei Ingham, Philip William Fujii, Hodaka Lee Kong Chian School of Medicine (LKCMedicine) Lee Kong Chian School of Medicine Embryos Ribozymes CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version 2016-12-09T08:45:02Z 2019-12-06T15:43:09Z 2016-12-09T08:45:02Z 2019-12-06T15:43:09Z 2016 Journal Article Lee, R. T. H., Ng, A. S. M., & Ingham, P. W. (2016). Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis. PLOS ONE, 11(11), e0166020-. 1932-6203 https://hdl.handle.net/10356/84342 http://hdl.handle.net/10220/41788 10.1371/journal.pone.0166020 en PLOS ONE © 2016 Lee et al. This is an open access article distributed underthe terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 12 p. application/pdf |
| spellingShingle | Embryos Ribozymes Lee, Raymond Teck Ho Ng, Ashley Shu Mei Ingham, Philip William Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis |
| title | Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis |
| title_full | Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis |
| title_fullStr | Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis |
| title_full_unstemmed | Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis |
| title_short | Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis |
| title_sort | ribozyme mediated grna generation for in vitro and in vivo crispr cas9 mutagenesis |
| topic | Embryos Ribozymes |
| url | https://hdl.handle.net/10356/84342 http://hdl.handle.net/10220/41788 |
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