Microalgae are versatile organisms capable of converting CO2, H2O and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.
We first generated a specific knockout of the CpFTSY gene that confers a smaller, or truncated, chlorophyll (Chl) antenna size of the photosystems. Previously, a null mutation of CpFTSY in C. reinhardtii caused drastically lower levels of the light-harvesting Chl binding proteins, lower Chl content and a higher Chl a to Chl b ratio than in wild type15. It was suggested that a smaller antenna size might prevent over absorption of sunlight, help greater light penetration of sunlight deeper into a high-density culture and thus contributing to greater productivity of mass culture under bright sunlight16. To this end, we carefully designed four small guide RNAs (sgRNAs) that would cause microhomology-driven frameshift mutations in the target gene using Cas-Designer (www.rgenome.net/cas-designer)17,18 (Supplementary Fig. 1). We then transfected RGEN-RNPs into C. reinhardtii cells (107 cells) by electroporation and streaked them out on a Petri dish (Fig. 1a).
We next applied RGEN-RNPs to block zeaxanthin (Zea) epoxidation and, thereby, to accumulate this xanthophyll in C. reinhardtii. Zea is a macular pigment of retina, which can prevent the development of chronic diseases such as age-related macular degeneration by filtering hazardous blue light and UV20. Blocking the epoxidation step from Zea to violaxanthin (Vio), catalyzed by the zeaxanthin epoxidase (ZEP), can lead to constitutive accumulation of Zea21,22 (Fig. 2a). Accordingly, we designed five sgRNAs in the ZEP locus (Supplementary Fig. 5) as described above and transfected each sgRNA with the purified Cas9 protein into C. reinhardtii cells. Targeted deep sequencing showed that indels were induced at a frequency of 0.46% (Supplementary Fig. 6). We picked several putative ZEP knockout cells by measuring the Chl fluorescence of the various colonies (Supplementary Fig. 7) and were able to obtain at least three ΔZEP mutants, confirmed by Sanger sequencing (Fig. 2b and Supplementary Fig. 8) and by measuring the amounts of Zea and Vio via HPLC (Supplementary Fig. 9). As expected, indel patterns were shown at the expected positions (Fig. 2b) and Zea were significantly increased more than ten times in ΔZEP mutants compared to the wild type, even under low light growth conditions (Fig. 2c).
How to cite this article: Baek, K. et al. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci. Rep. 6, 30620; doi: 10.1038/srep30620 (2016).
Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.
In addition to the generation of knock-in mice carrying complex gene cassettes, our method can be directly applied to the generation of knockout mice  and knock-in mice carrying single nucleotide substitutions with oligo DNA donors [5,7,8], as well as to other species [39-41] and cultured cells [22,28], and in vivo genome editing in adult animals . Taken together, our streamlined cloning-free CRISPR/Cas-mediated in vivo genome editing system enables the highly efficient and extremely convenient one-step generation of knock-in mice carrying functional gene cassettes.
Cas9 protein and chemically synthesized crRNA and tracrRNA enabled cloning-free CRISPR/Cas system without CRISPR vector construction, cellular experiments for evaluation of the digestion activity of CRISPR/Cas, and in vitro RNA transcription. By the direct nuclear delivery of Cas9 protein complex combined with dual RNAs into one-cell mouse zygotes, knock-in mice carrying functional cassette were generated with extreme high efficiency, which could not be achieved by conventional mRNA pronuclear injection or Cas9 protein injection combined with commonly used sgRNA. Taken together, our streamlined cloning-free CRISPR/Cas-mediated in vivo genome editing system provides the highly efficient and extremely convenient one-step generation of knockout and knock-in animals, leading to acceleration of in vivo functional genomic research.
Cas9 mRNA and Actb-sgRNA were prepared as described previously . Cas9 and Actb-sgRNA were PCR amplified from Actb-pX330 with T7 promoter-attached primers (Table S6 in Additional file 1). T7-Cas9 and T7-Actb-sgRNA PCR products were purified with a PCR Purification Kit (Qiagen) and used as the template for in vitro transcription using a mMESSAGE mMACHINE T7 ULTRA Kit (Life Technologies) and MEGAshortscript T7 Kit (Life Technologies). Cas9 mRNA and Actb-sgRNA were purified with a MEGAclear Kit (Life Technologies) and eluted with Nuclease-free water (Life Technologies). The quality of RNAs was analyzed using a NanoDrop (Thermo Scientific, Waltham, MA, USA) and Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Effects of SlARF4 knockout on resistance to water deficit, morphology, and transpirational water loss in tomato plants. (A) Degree of curliness of 2-month-old tomato leaves; (B) Thick stem of two-month-old tomato plants. (C) Rate of water loss from small leaves isolated from 2-month-old tomato plants at room temperature. (D) Rate of water loss from mature leaves excised from 2-month-old tomato plants at room temperature. (E) Effect of water deficit on wild-type (WT, left) and arf4 mutant (right) tomato plants. Data was means ± SE of three independent biological replicates. Different letters (a, b) presented significant difference at level set p < 0.05.
Plant water status is another important indicator for evaluating plant resistance to water deficit . In the experiments reported here, the rate of water loss in arf4 leaves was lower than that recorded for the WT leaves. On the one hand, it benefited from leaf curling, which reduced transpiration, whereas at the same time, the stomata did not close completely, thereby maintaining transpiration activity; on the other hand, compared with the WT plants, water loss was slow, which was also reflected by the lower free water content. The rate of water loss in arf4 large leaves with petioles was higher than that of the WT leaves, which was closely related to the more developed xylem in arf4, whereby the latter was able to hold more water and, thus, maintain an enduring hydrated status .
ParkUSA, a Northwest Pipe Company, provides innovative water, wastewater, and stormwater equipment and technology for infrastructure and built environments. Our design and manufacturing capabilities offer client focused solutions to meet the growing demands and regulatory requirements of the water industry.
Recombinant proteins manufactured in E. coli are inherently contaminated with endotoxin. In light of the enormous diversity of recombinant proteins expressed in E. coli, none of the purification methods are universally applicable to significantly reduce the endotoxin content. Instead of removing endotoxin from the protein samples, the work presented here demonstrates that it is now possible to eliminate endotoxin at the source by producing proteins in an endotoxin-free environment using LPS-free derivatives of E. coli K-12 and BL21 (DE3) strains. These non-conditional mutants clearly lack hTLR4/MD-2 signaling pathway agonists yet can retain viability through predominantly synthesizing the tetraacylated, endotoxically inactive lipid A precursor lipid IVA. The design of the strains also prohibits them from easily regaining the potential to synthesize normal LPS or endotoxically active lipid IVA derivatives through acquiring mutations. This has been accomplished by incorporating a total of seven non-reverting genetic deletions that disrupt Kdo biosynthesis and prevent lipid IVA from being modified with enzymes of both the constitutive and the regulated LPS pathway, while the compensating mutations msbA52 and msbA148 enable the E. coli K-12- and BL21 (DE3)-derived cells to maintain viability, respectively. The derivation of E. coli strains with dramatically modified LPS affords the unique opportunity to produce endotoxin-free recombinant proteins suitable for downstream experiments with human cells. These strains allow researchers to save time-consuming cleanup steps that may affect yield and functionality of the end product. 2b1af7f3a8