Repositorium
Journal Article / 2016
Shin, Sung-Eun; Lim, Jong-Min; Koh, Hyun Gi; Kim, Eun Kyung; Kang, Nam Kyu; Jeon, Seungjib; Kwon, Sohee; Shin, Won-Sub; Lee, Bongsoo; Hwangbo, Kwon; Kim, Jungeun; Ye, Sung Hyeok; Yun, Jae-Young; Seo, Hogyun; Oh, Hee-Mock; Kim, Kyung-Jin; Kim, Jin-Soo; Jeong, Won-Joong; Chang, Yong Keun; Jeong, Byeong-Ryool
Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including “safe harboring” techniques shown in other organisms.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
446 |
Jeong, Won-Joong; Chang, Yong Keun; Jeong, Byeong-ryool South Korea |
Chlamydomonas reinhardtii |
CRISPR/Cas9 CpSRP43 |
encodes chloroplast SPR43 SDN1 |
Basic research Basic research |
447 |
Jeong, Won-Joong; Chang, Yong Keun; Jeong, Byeong-ryool South Korea |
Chlamydomonas reinhardtii |
CRISPR/Cas9 ChlM |
encodes Mg-protoporphyrin IX S-adenosyl methionine O-methyl transferase SDN1 |
Basic research Basic research |
448 |
Jeong, Won-Joong; Chang, Yong Keun; Jeong, Byeong-ryool South Korea |
Chlamydomonas reinhardtii |
CRISPR/Cas9 MAA7 |
encodes the beta subunit of trytophan synthase SDN1 |
Basic research Basic research |