An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
08 04 2021
08 04 2021
Historique:
received:
12
12
2020
accepted:
05
03
2021
entrez:
9
4
2021
pubmed:
10
4
2021
medline:
10
11
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 33833247
doi: 10.1038/s41598-021-86549-9
pii: 10.1038/s41598-021-86549-9
pmc: PMC8032657
doi:
Substances chimiques
DNA, Plant
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
7695Références
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