Targeted mutagenesis of peroxisomal protein phosphatases using CRISPR-Cas9 approach
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The newest development in the techniques of gene editing is the clustered regularly interspaced short palindromic repeat - associated protein9 (CRISPR-Cas9). This is a complex consisting of single-guide RNA (sgRNA) and a Cas9 protein. The sgRNA can, based on designing of specific spacers, recognize specific target sites in the DNA leading to double stranded breaks (DBS) through cutting by Cas9. In this study, the establishment of CRSISPR/Cas9 editing for stable plant transgenic lines was aimed. Selection of suitable plasmids harboring Cas9 encoding gene, and genes encoding sgRNA components was accomplished. The selection of vectors depended also on choosing of optimum promoter, for example using the egg cell specific promotor EC1.1 promoter fused with an EC1.2 enhancer in Arabidopsis thaliana (Arabidopsis) and the Cauliflower mosaic virus (CaMV) 35S promotor in Solanum lycopersicum (S. lycopersicum). The CRISPR/Cas9 method was used in order to knockout peroxisomal protein phosphatases in Arabidopsis, and the putative regulator of protein phosphatase 4 (PSY2L) in tomato. Two predicted spacers were used for generating two sgRNAs in order to direct Cas9 to two different targets in the genome of Arabidopsis purple acid phosphatase 7 (PAP7), Pol-like phosphatase (PLL2, PLL3, PLL3/PLL2 (two different combinations)), and two variants for putative PSY2L in tomato. Golden gate cloning was used to clone the selected spacers, and the obtained pCAMBIA-based binary vectors including cloned Cas9 and constructed sgRNAs were transformed to Agrobacterium tumefaciens (Agrobacterium) GV3101. Subsequently, Arabidopsis plants were transformed with these vectors, and seeds were screened for successful transformations. Genomes for the T1 generated plants were analyzed by T7 endonuclease 1 assay (T7E1), and only heterozygote plants could be detected. We were able to obtain 10/10 heterozygote PAP7 mutants, 3/5 heterozygote PLL3 mutants, 2/5 heterozygote PLL2 mutants and 1/10 PLL3/PLL2 double mutants for Arabidopsis. T2 generations was also screened on selectable markers, and representative plants were examined phenotypically. Further analysis will be needed to distinguish between wild type and homozygote plants using T7E1 assay and/or by sequencing. Moreover, the constructed pCAMBIA-based binary vectors and pGreen-based binary vectors for editing PSY2L in tomato, and peroxisomal phosphatases in Arabidopsis are successfully cloned.
Master's thesis in Biological chemistry