Versatile application of the CRISPR-Cas system to various organisms

Abstract

CRISPR-Cas is an adaptive immune system in bacteria and archaea. It specifically finds target through a complementary DNA-RNA hybridization and removes target DNA or RNA. A genome engineering has advanced explosively due to an introduction of CRISPR-Cas, moreover numerous studies have been accomplishing with CRISPR-Cas in most of scientific fields. Studies in below were a few instances of various applications using CRISPR-Cas. The first study developed a cancer diagnosis method in a liquid biopsy using CRISPR-Cas. CUT (CRISPR-mediated, Ultrasensitive detection of Target DNA)-PCR can detect the extremely small amounts of tumor DNA fragments among the much more abundant wild-type DNA fragments by specifically eliminating the wild-type sequences with CRISPR endonucleases and enriching tumor DNA. I computed that by using various orthologonal CRISPR endonucleases, the CUT-PCR method would be applicable to 80% of known cancer-linked substitution mutations. I further verified that CUT-PCR together with targeted deep sequencing enables detection of a broad range of oncogenes with high sensitivity Second study showed a generation of Crltd mutants by DNA-free ribonucleoprotein delivery in microalgae, Chlamydomonas reinhardtii. Crltd mutants which were generated using CRISPR-Cas had a pale-yellow coloration and reduced chlorophyll and PSI complex contents than wild type. As a results, it was supposed that CrLTD involved in a trafficking of LHCP to thylakoid membranes in a chloroplast. An analysis of off-target mutations in Crltd1 mutant confirmed that there were no unwanted mutations by an off-targeting. This study also optimized the method of the ribonucleoprotein delivery in C. reinhardtii, thus made much easy the generation of mutants by CRISPR-Cas. Finally, I tried a targeted DNA methylation using inactive Cas9 protein and DME protein which is a plant-specific DNA demethylase. A catalytic domain of DME fused to inactive Cas9, it was introduced to human cells and plants to induce a DNA demethylation at specific regions. In human cells, no significant changes were observed in the target region, KLF4 CpG island. In plant, FWA promoter region were targeted for generating fwa epi-mutants. Some of transgenic plants showed late flowering and vegetative axillary meristem, but DNA methylation levels of these plants were comparable to wild type. In addition, phenotypes of transgenic plants were not inherited in a next generation. It proved unsuccessful to develop an epi-genetic tool using dCas9-DME as present, it is expected that the tool could be improved by further experiments for an optimization. The epi-genome engineering has an advantage of changing phenotypes without DNA information changes. Therefore, dCas9-DME seems to have a potential as a tool for the epi-genome engineering.