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Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins
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Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins
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Journal Article
Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins
2016
Overview
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
×
hybrida
protoplast system.
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 site-directed 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.
Publisher
Springer Berlin Heidelberg,Springer Nature B.V
Subject
/ Biomedical and Life Sciences
/ breeding
/ CRISPR-Cas Systems - genetics
/ DNA
/ genes
/ Genetic Engineering - methods
/ High-Throughput Nucleotide Sequencing
/ loci
/ Mutagenesis, Site-Directed - methods
/ Mutation
/ Petunia
/ Recombinant Proteins - genetics
/ Recombinant Proteins - metabolism
/ Ribonucleoproteins - genetics
/ Ribonucleoproteins - metabolism
/ RNA
