CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

Sung Eun Shin, Jong Min Lim, Hyun Gi Koh, Eun Kyung Kim, Nam Kyu Kang, Seungjib Jeon, Sohee Kwon, Won Sub Shin, Bongsoo Lee, Kwon Hwangbo, Jungeun Kim, Sung Hyeok Ye, Jae Young Yun, Hogyun Seo, Hee Mock Oh, Kyung Jin Kim, Jin Soo Kim, Won Joong Jeong, Yong Keun Chang, Byeong Ryool Jeong

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286 Scopus citations


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 Cas9-induced 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.

Original languageEnglish
Article number27810
JournalScientific Reports
StatePublished - 13 Jun 2016


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