To knock out SH3RF2, 7.5 [micro]L of Lipofectamine 3000 Reagent was diluted in 250 [micro]L of OPTI-MEM (Invitrogen, USA), and 2.5 [micro]g each of the Cas9-green fluorescent protein (GFP) co-expression plasmids (Sigma-Aldrich, USA) and SH3RF2 gRNA
were mixed with Lipofectamine P3000 Reagent in 250 [micro]L of OPTIMEM at room temperature.
The chimeric gRNA
containing 20 nucleotide target sequence and 80 nucleotide scaffold sequence was commercially synthesized (e-oligos, Gene LinkTM NY, USA) to target NbPDS gene.
The GeneArt CRISPR Nuclease Vector Kits provide reporter and enrichment tags for tracking gene editing experiments and could be utilised for cloning and re-use of confirmed gRNA
The (-) viral genomic RNA, termed gRNA
, is a template for synthesis of mRNA and the intermediate (+) viral genomic RNA (termed cRNA in this study); the latter was used as the template for gRNA
synthesis [7, 26, 27].
Production and persistence of gRNA
and sgRNA were examined in permissive and nonpermissive cells.
In the mechanism his group proposes, short gRNA
molecules form a double-stranded complex with certain mRNA molecules.
(20 bp and 19 bp target sequences of left and right gRNA
, respectively) were designed with +7 bp offset (Figure 1B).
The SureGuide gRNA
synthesis kit provides highly efficient synthesis with an optimised purification step in a streamlined protocol.
A strand of gRNA
made of 20 nucleotides was constructed by the researches which was used as the target system of the tool.
CRISPR (clustered, regularly interspaced short palindromic repeats) is a novel RNA-guided gene-editing technology that makes use of a bacterially derived protein (Cas9) and a synthetic guide RNA (gRNA
) to introduce a break at a certain location within the genome.