Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing.
As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method.
Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka, Fukuoka 814-0180, Japan.
Masatora
M
Fukuda
Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka, Fukuoka 814-0180, Japan.
Hiromitsu
H
Umeno
Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka, Fukuoka 814-0180, Japan.
Kanako
K
Nose
Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka, Fukuoka 814-0180, Japan.
Azusa
A
Nishitarumizu
Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka, Fukuoka 814-0180, Japan.
Ryoma
R
Noguchi
Department of Earth System Science, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka, Fukuoka, 814-0180, Japan.
Hiroyuki
H
Nakagawa
D000241:Adenosine / Q000378:metabolism*; D000243:Adenosine Deaminase / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007288:Inosine / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D000072417:Protein Domains; D017393:RNA Editing / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems
D000241:Adenosine; D000243:Adenosine Deaminase; D003062:Codon; D007288:Inosine; D016601:RNA-Binding Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C567955:ADARB1 protein, human