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Sci Rep. 2016 Jun 13;6:27810. doi: 10.1038/srep27810.

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

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Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
Advanced Biomass R&D Center (ABC), KAIST, Daejeon 34141, Republic of Korea.
Department of Biological Science, Chungnam National University (CNU), Daejeon 34134, Republic of Korea.
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea.
Department of Chemistry, Seoul National University (SNU), Seoul 08826, Republic of Korea.
Basic science, IBS school, Korea University of Science and Technology (UST), Seoul 08826, Republic of Korea.
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University (KNU), Daegu 41566, Republic of Korea.


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.

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