Transient expression-based CRISPR/Cas9 system for manipulation of tall fescue SGR gene

  A clustered regularly interspaced short palindromic repeat (CRISPR) is one of the precise genome editing techniques. In present experiment guide RNA associated to the tall fescue SGR gene (FaSGR) joint to the Cas9 protein in CRISPR system so as to target SGR in tall fescue ((Festuca arundinacea 'Jaguar ′) leaves. Materials and methods In order to construct programmable CRISPR/Cas9 system for targeting FaSGR, 20 nucleotides of exon originated FaSGR gene were selected. This 20 bp firstly integrated into pEn/Chimera vector followed by LR reaction (gateway system) in which aforementioned segment transferred into the ccdB frame of expression vector. Sequenced and PCR certified vector by gene specific primers (GSPs) then inserted to the GV 3101 strain of Agrobacterium tumefaciens. For construction of overexpression vector (OE) of SGR gene, full length of FaSGR was isolated from tall fescue leaf through gene specific primers and after cloning in E-Coli integrated into pB2WG7 vector using Gateway technology. In second step CRISPR construct and overexpression pB2WG7 vector harboring full length of FaSGR, co-transformed into tobacco leaves through agroinfiltration method. DNA extracted from agro-infected area and sequenced by gene specific primers. In other experiment CRISPR /Cas9 construct transiently and directly expressed in young mature tall fescue leaves subjected to abiotic stress. Polymerase chain reaction and DNA sequencing strongly support guide RNA order in two vectors. Transient co-expression of two separate vectors in tobacco leaves after receiving DNA extraction and sequencing data, vividly showed, although the segment sequence and order of guide RNA is fine but no deletion in FaSGR gene was observed by applying CRISPR/Cas9 system. However in second experiment transient expression of CRISPR/CAS9 in tall fescue leaves which having natural FaSGR gene under vacuum pressure surprisingly suppress SGR expression whereupon improve tall fescue leaf color quality compared to control leaves. Gateway technology which has been used in this experiment greatly improved preciseness of CRISPER/Cas9 construct design and shorten times in contrast to that of restriction enzymes method. On model plant tobacco CRISPR/Cas9 was not successful in order to cut FaSGR gene on target site. This likely can be due either to the heterologous expression of this gene in untargeted organisms or different promoter governs genes. However, when we applied this transient expression-based genome editing system on tall fescue leaves under vacuum, we clearly observed FaSGR inactivated by CRISPR/Cas9, as a result tall fescue leaves stayed much greener under salt and heat stress than control.