Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins

Abstract

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.

Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes

“…However, in plants, the presence of a cell wall makes it impossible to use transfection or electroporation for nucleic acid and/or protein delivery. Recently, plant protoplasts, generated by the removal of this cell wall using enzymatic digestion, have been successfully used for RNP delivery and genome editing in a variety of plants such as tobacco, Arabidopsis , lettuce, rice and Petunia 1516. However, for the majority of monocotyledon species, including major crops like maize, wheat, rice, barley and sorghum, regeneration of plants from protoplasts remains either unattainable or inefficient1718.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…The CRISPR-Cas system can be stably or transiently transfected into the plant cells as nucleic acids (plasmids or DNA fragments) [6,73] or pre-assembled ribonucleoprotein complexes (RNPs) [74][75][76].…”

“…Using Cas9 RNA or protein presents the advantage of not having to work on optimizing expression by defining sequences codon-optimized for the host plant and selecting appropriate promoters. Preassembled complexes of purified Cas9 protein and guide RNA (RNPs) were transfected into protoplasts of Arabidopsis thaliana, Nicotiana attenuata, Petunia x hybrida, grapevine, apple, lettuce and rice [74,76,95] and bombarded into immature embryos of bread and durum wheat [73,96], and maize [75]. In all cases, RNPs showed good cleavage efficiency (4-46%), with (when tested) no detectable mosaicism, suggesting a very early action of the nuclease before the first cell division [74,76].…”

Targeted genome editing, an alternative tool for trait improvement in horticultural crops

“…CRISPR/Cas9-mediated genome editing can be achieved by either transfecting the cells with a plasmid encoding CRISPR-associated protein 9 (CAS9) and single-guide RNA (sgRNA) or by direct delivery of pre-complexed purified CAS9 protein along with in vitrotranscribed sgRNA . The CRISPR/Cas9 system has been used to edit the genomes of many plants (Jiang et al, 2013;Hyun et al, 2015;Subburaj et al, 2016b), animals , bacteria (Jinek et al, 2012), and yeast .…”