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...
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Nature Portfolio
2021
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oai:doaj.org-article:cf7ad386493a4ad5925bfe5e9dd796132021-12-02T18:15:33ZAn improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue10.1038/s41598-021-86549-92045-2322https://doaj.org/article/cf7ad386493a4ad5925bfe5e9dd796132021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-86549-9https://doaj.org/toc/2045-2322Abstract 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.Kyle MillerAlan L. EggenbergerKeunsub LeeFei LiuMinjeong KangMadison DrentAndrew RubaTyler KirschtKan WangShan JiangNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021) |
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DOAJ |
| collection |
DOAJ |
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EN |
| topic |
Medicine R Science Q |
| spellingShingle |
Medicine R Science Q 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 |
| 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. |
| format |
article |
| 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 |
| title |
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_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_sort |
improved biolistic delivery and analysis method for evaluation of dna and crispr-cas delivery efficacy in plant tissue |
| publisher |
Nature Portfolio |
| publishDate |
2021 |
| url |
https://doaj.org/article/cf7ad386493a4ad5925bfe5e9dd79613 |
| work_keys_str_mv |
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