An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue
Abstract Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2021-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-86549-9 |
| _version_ | 1818742916299882496 |
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| author | Kyle Miller Alan L. Eggenberger Keunsub Lee Fei Liu Minjeong Kang Madison Drent Andrew Ruba Tyler Kirscht Kan Wang Shan Jiang |
| author_facet | Kyle Miller Alan L. Eggenberger Keunsub Lee Fei Liu Minjeong Kang Madison Drent Andrew Ruba Tyler Kirscht Kan Wang Shan Jiang |
| author_sort | Kyle Miller |
| collection | DOAJ |
| description | Abstract Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT-2020. In addition, the same approach is applied to test the efficacy of multiple gRNAs for CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues. |
| first_indexed | 2024-12-18T02:20:07Z |
| format | Article |
| id | doaj.art-cf7ad386493a4ad5925bfe5e9dd79613 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-18T02:20:07Z |
| publishDate | 2021-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-cf7ad386493a4ad5925bfe5e9dd796132022-12-21T21:24:17ZengNature PortfolioScientific Reports2045-23222021-04-0111111110.1038/s41598-021-86549-9An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissueKyle Miller0Alan L. Eggenberger1Keunsub Lee2Fei Liu3Minjeong Kang4Madison Drent5Andrew Ruba6Tyler Kirscht7Kan Wang8Shan Jiang9Department of Materials Science and Engineering, Iowa State UniversityDepartment of Materials Science and Engineering, Iowa State UniversityCrop Bioengineering Center, Iowa State UniversityDepartment of Materials Science and Engineering, Iowa State UniversityCrop Bioengineering Center, Iowa State UniversityDepartment of Materials Science and Engineering, Iowa State UniversityDepartment of Materials Science and Engineering, Iowa State UniversityDepartment of Materials Science and Engineering, Iowa State UniversityCrop Bioengineering Center, Iowa State UniversityDepartment of Materials Science and Engineering, Iowa State UniversityAbstract Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT-2020. In addition, the same approach is applied to test the efficacy of multiple gRNAs for CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.https://doi.org/10.1038/s41598-021-86549-9 |
| spellingShingle | Kyle Miller Alan L. Eggenberger Keunsub Lee Fei Liu Minjeong Kang Madison Drent Andrew Ruba Tyler Kirscht Kan Wang Shan Jiang An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue Scientific Reports |
| title | An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue |
| title_full | An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue |
| title_fullStr | An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue |
| title_full_unstemmed | An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue |
| title_short | An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue |
| title_sort | improved biolistic delivery and analysis method for evaluation of dna and crispr cas delivery efficacy in plant tissue |
| url | https://doi.org/10.1038/s41598-021-86549-9 |
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