期刊
BMC BIOTECHNOLOGY
卷 19, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s12896-019-0530-x
关键词
Precise genetic editing; Genome engineering; CRISPR; Cas9; Protoplasting; Fluorescence activated cell sorting; Mutation enrichment; Nicotiana benthamiana
资金
- Danish Councils for Strategic and Independent Research [12-125709, 12-131859]
- Danish National Research Foundation [DNRF107]
- Copenhagen University Excellence Program for Interdisciplinary Research (CDO2016)
- Villum Foundation [00017489]
BackgroundCRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand.ResultsIn this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3-5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations.ConclusionsFACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.
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