Improvement of Chloroplast Transformation Using CRISPR/Cas9

Abstract

Chloroplasts are organelles that contain genetic materials (DNA) in higher plant cells. The special genetic characteristics of chloroplasts mean that plasmid transformation has important research value, so it has become an important research direction second to nuclear transformation. Although the techniques of chloroplast genome modification have been successfully applied in tobacco and extended to other high plants, there are still many limitations. Exogenous genes are integrated into the chloroplast genome through homologous recombination. Therefore, the low efficiency of homologous recombination directly limits transformation efficiency. Gene editing with fixed-point cutting function and DNA damage repair mechanism may effectively improve the efficiency. In the present study, we aimed to use CRISPR/Cas9 to cut the site between two homologous recombinant fragments in chloroplast transformation to improve the efficiency by activating the DNA damage repair mechanism. The Cas9 gene and gRNA were added to the chloroplast transformation system of tobacco by co-transformation or integration into a transformation vector. The acquired resistant plants were screened by multiple selection of spectinomycin and chloroplast DNA was isolated for molecular detection by PCR. The results showed that the efficiency of chloroplast transformation increased by 6–10 times with the addition of gene editing technology. Although the transformation efficiency was still far below the level of nuclear transformation, this study may help to increase the efficiency of the plant chloroplast transformation system, and expand the types of plant receptors.

The Search of a Molecular “Swiss Knife” for Chloroplast Genomic Editing

“…Genes encoding editing tools were delivered into chloroplasts either in the co-transformation mode or as part of an expression cassette along with target genes and a selectable marker in a single vector. The results showed that the efficiency of chloroplast transformation with this supplement increased six-to ten-fold [40].…”

“…These plasmids were shown to be able to autonomously replicate, express and produce Cas9, gRNA and donor DNA within the chloroplasts [39]. By using bioballistics, plasmids that encode editing tools were also successfully delivered into the chloroplasts of tobacco leaves, where they were effectively expressed, increasing the frequency of transgene integration [40].…”

Three Parts of the Plant Genome: On the Way to Success in the Production of Recombinant Proteins

“…An analogous gene construct with the genome-editing system has also been employed to transform tobacco chloroplasts. The authors demonstrated that the targeted DSBs produced by the Cas9/sgRNA complex raise the rate of chloroplast transformation by 6–10-fold [ 170 ].…”