SITE-DIRECTED MUTAGENESIS IN PETUNIA X HYBRIDA PROTOPLAST SYSTEM USING DIRECT DELIVERY OF PURIFIED RECOMBINANT CAS9 RIBONUCLEOPROTEINS

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Title
SITE-DIRECTED MUTAGENESIS IN PETUNIA X HYBRIDA PROTOPLAST SYSTEM USING DIRECT DELIVERY OF PURIFIED RECOMBINANT CAS9 RIBONUCLEOPROTEINS
Author(s)
Subburaj S; Chung SJ; Choongil Lee; Seuk-Min Ryu; Kim DH; Jin Soo Kim; Bae S; Lee GJ
Publication Date
2016-07
Journal
PLANT CELL REPORTS, v.35, no.7, pp.1535 - 1544
Publisher
SPRINGER
Abstract
Key message Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia 3 hybrida protoplast system. Abstract The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for sitedirected mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3–17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems. Springer-Verlag Berlin Heidelberg 2016
URI
https://pr.ibs.re.kr/handle/8788114/5806
ISSN
0721-7714
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > Journal Papers (저널논문)
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