CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.
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.
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Sung-Eun
SE
Shin
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
Jong-Min
JM
Lim
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Hyun Gi
HG
Koh
Advanced Biomass R&D Center (ABC), KAIST, Daejeon 34141, Republic of Korea.
Eun Kyung
EK
Kim
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Nam Kyu
NK
Kang
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Seungjib
S
Jeon
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Sohee
S
Kwon
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Won-Sub
WS
Shin
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Bongsoo
B
Lee
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.|Department of Biological Science, Chungnam National University (CNU), Daejeon 34134, Republic of Korea.
Kwon
K
Hwangbo
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.
Jungeun
J
Kim
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea.|Basic science, IBS school, Korea University of Science and Technology (UST), Seoul 08826, Republic of Korea.
Sung Hyeok
SH
Ye
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea.
Jae-Young
JY
Yun
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University (KNU), Daegu 41566, Republic of Korea.
Hogyun
H
Seo
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
Hee-Mock
HM
Oh
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University (KNU), Daegu 41566, Republic of Korea.
Kyung-Jin
KJ
Kim
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.
Jin-Soo
JS
Kim
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
Won-Joong
WJ
Jeong
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.|Advanced Biomass R&D Center (ABC), KAIST, Daejeon 34141, Republic of Korea.
Yong Keun
YK
Chang
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Byeong-Ryool
BR
Jeong
D020418:Algal Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002734:Chlorophyll / Q000737:chemistry; D059766:DNA End-Joining Repair / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D056426:Genetic Loci; D016296:Mutagenesis; D030821:Plants, Genetically Modified / Q000235:genetics; D034622:RNA Interference; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014367:Tryptophan Synthase / Q000737:chemistry / Q000145:classification / Q000235:genetics; D000073336:Whole Genome Sequencing
D020418:Algal Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D002734:Chlorophyll; D014367:Tryptophan Synthase