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

Seungjae Shin ,Sung Hyeok Ye,Jae-Young Yun,Jin-Soo Kim,Bongsoo Lee,Hyun Gi Koh,Won-Joong Jeong,Yong Keun Chang,Hogyun Seo,Sohee Kwon,Kwon Hwangbo,Jong‐Min Lim ,Min Jung Kim ,Nak Heon Kang ,Sang Goo Jeon ,Kyungjin Kim ,Jung-Eun Kim ,Wonsub Shin ,Hee‐Mock Oh ,Byeong‐Ryool Jeong

doi:10.1038/srep27810

Abstract

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.

Highlights

Microalgae have emerged as the third generation biofuel feedstocks that are excellent renewable and sustainable energy sources for the future
A new system based on components of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), has become very popular in disrupting a gene due to its simplicity, improved specificity and higher versatility compared to zinc-finger nuclease (ZFN) and TALEN24,25
We successfully generated a sizable number of CRISPR/Cas9-induced mutations at the MAA7, CpSRP43 and ChlM loci by delivering Cas[9] RNP

Summary

Introduction

Microalgae have emerged as the third generation biofuel feedstocks that are excellent renewable and sustainable energy sources for the future Their use would allow us to replace the current fossil fuels that have been blamed for serious environmental problems, including pollution and global warming[1]. Genes can be down-regulated by RNA silencing techniques involving RNA interference (RNAi) or artificial microRNAs (amiRNA)[13,14,15,16,17] These techniques can be used to study essential genes, for which recessive mutations are lethal, in haploid microalgae; these methods are often limited by low efficiency, non-specific targeting and silencing of the RNA constructs[9,18]. One such report exists for Chlamydomonas, and extremely low targeting efficiency was observed, possibly reflecting toxicity induced by vector-driven Cas[9] expression[29]

Methods

Results

Conclusion