Abstract:
BACKGROUND:CRISPR/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. RESULTS:In 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. CONCLUSIONS:FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.
journal_name
BMC Biotechnoljournal_title
BMC biotechnologyauthors
Petersen BL,Möller SR,Mravec J,Jørgensen B,Christensen M,Liu Y,Wandall HH,Bennett EP,Yang Zdoi
10.1186/s12896-019-0530-xsubject
Has Abstractpub_date
2019-06-17 00:00:00pages
36issue
1issn
1472-6750pii
10.1186/s12896-019-0530-xjournal_volume
19pub_type
杂志文章abstract:BACKGROUND:The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody - directed enzyme-prodrug therapy (GDEPT/ADEPT) a...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-8-68
更新日期:2008-09-10 00:00:00
abstract:BACKGROUND:Transformed cells of Escherichia coli DH5-alpha with pGFPuv, induced by IPTG (isopropyl-beta-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-p...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/1472-6750-2-7
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journal_title:BMC biotechnology
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journal_title:BMC biotechnology
pub_type: 杂志文章
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journal_title:BMC biotechnology
pub_type: 杂志文章
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journal_title:BMC biotechnology
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journal_title:BMC biotechnology
pub_type: 杂志文章
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更新日期:2012-05-03 00:00:00
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journal_title:BMC biotechnology
pub_type: 杂志文章
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更新日期:2009-05-14 00:00:00
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journal_title:BMC biotechnology
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更新日期:2007-06-29 00:00:00
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journal_title:BMC biotechnology
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更新日期:2015-06-04 00:00:00
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更新日期:2015-02-13 00:00:00
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更新日期:2010-05-13 00:00:00
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更新日期:2007-02-26 00:00:00
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更新日期:2014-12-16 00:00:00
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更新日期:2013-02-26 00:00:00
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更新日期:2014-01-30 00:00:00
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