Cas9關聯核糖核蛋白複合體(Cas9-RNP)是一種新型態的常間迴文重複序列叢集(CRISPR)的應用系統，不同於以往的送入質體使宿主自表達之方式，此核酸酶蛋白具有基因編輯功能而被直接送入宿主細胞內作用，且能達成無痕基因編輯的用途。本研究分為三部分，首先建立大腸桿菌BL21(DE3)的雙質體系統共表達Cas9蛋白和單導向RNA (sgRNA)，其次進行體外(in-vitro)基因剪切功能分析，最後測試微藻的無痕基因編輯，提昇細胞代謝產物的生成。
首先建構具有高拷貝數的通用質體pSG-RSF，插入靶點在1,5-二磷酸核酮糖羧化酶之基因(rbcL)的單導向RNA，在大腸桿菌合成的Cas9-RNP經由鎳金屬親和層析純化，得知N-末的His-標籤蛋白融合比在C-端融合更有助於提高純化回收率，從而降低成本；且平均於每50毫升培養菌液中生產200微克之Cas9-RNP。利用PCR擴增萊茵依藻基因組的rbcL基因，試驗Cas9-RNP的基因剪切能力，結果分析發現反應在1、2、4、8小時的活性不變，且活性在21天內穩定，另外本研究亦首次探討可增加表達水平的基因調控元件“RiboJ”被利用於CRISPR系統中以增加Cas9蛋白表達；然而Cas9因基因片段達4107 bp而蛋白無法增加，其他基因片段在600 bp 到 2700 bp的結果均提高，顯示被調控之基因受限於一定長度，且RiboJ不適用在弱啟動子驅動Cas9表現匣。最後，我們以Cas9-RNP介導CC400之八氫番茄紅素合成酶基因 (psy)，從而調控類胡蘿蔔素之代謝途徑。最佳的電穿條件為使用單次180伏特之穿孔脈衝與四次20伏特之傳遞脈衝，以20微克之Cas9-RNP送入衣藻，經液體加入100 mg/L的氨苄青黴素青黴素繼代培養，經由基因編輯的表現型藻株顏色變淺色，而在生長表現方面與野生型無明顯差異，由此可推測Cas9-RNP確實影響了依藻色素生成的代謝路徑。再由高效液相層析分析類胡蘿蔔素在依藻體內之累積，發現其色譜圖顯示之偵測峰的形式不同於野生型，因此驗證了Cas9-RNP編輯psy基因後迫使依藻之類胡蘿蔔素的代謝通量產生改變。闡明了可編程之Cas9-RNP適用於CC400的無痕基因組編輯。Cas9-RNP會自行降解而無法在宿主細胞內長時間作用，將可解決Cas9蛋白因長時間表達產生內毒素並降低脫靶效應。

Cas9-associated ribonucleoproteins (Cas9-RNPs) is a novel application of CRISPR system and can achieve seamless DNA editing, which different from previous strategy, that delivering nuclease of gene-editing function into host cells instead of expressed by itself then functioning. Three part are mainly contained in this study. First, establishment of a dual plasmid system which co-express Cas9 protein and single guide RNA (sgRNA). Second, analysis of gene cleavage functions by in vitro test. Finally, mediating Cas9-RNP into microalgae Chlamydomonas reinhardtii (C. reinhardtii) CC400 for seamless gene editing and improving production of cell metabolites by in vivo test.
First of all, a common-used plasmid pSG-RSF with high copy number was constructed. Afterwards, sgRNA which target on Ribulose-1,5-bisphosphate carboxylase (rbcL) gene was inserted into the pSG-RSF. It was ascertained that His-tag protein fusion at N-terminus is more helpful in increasing recovery rate of purification than fusion at C-terminus as well to decrease production cost. Average of 200 g Cas9-RNP was produced from each 50 mL E. coli cultured medium after purification by nickel ion affinity chromatography. Hereafter, Cas9-RNP capability of gene cleavage was validated by in vitro test with utilization of amplified fragment that PCR from C. reinhardtii CC400 genome. Analysis result represented that activity performed stable when reaction times at 1, 2, 4, 8 hours and within produced 21 days. Additionally, a gene element ‘RiboJ’ which can enhance expression level is first investigated in this study for improving Cas9 protein expression in CRISPR system. However, negative result was observed for enhancing protein expression due to length up to 4107 bp of Cas9 gene fragment as other gene fragments from about 600 bp to 2700 bp were enhanced, in contrast. Results illustrated that open reading frame (ORF) of regulated genes are limited below certain length and RiboJ is not suitable for enhancing Cas9 express cassette driven by a weak promoter. Finally, we introduced Cas9-RNP that mediate phytoene synthase (psy) gene in C. reinhardtii CC400 to regulate metabolic flux in carotenoid pathway under the optimized electroporation conditions as a 180 V poring pulse followed by four 20 V transfer pulse with 20 g of Cas9-RNP added. After cultivated in liquid medium with 100 mg/L ampicillin, phenotype of CC400 strain through gene editing represented light-green color which represented no distinction on cell growth compare to wild type (WT) strain. It can be speculated that Cas9-RNP does affect the metabolic pathway of algal pigment production. Different form of detection peak from WT has been discovered after analysis by high-performance liquid chromatography (HPLC). Therefore, it was validated that alteration of carotenoids metabolic flux forced by psy gene editing with Cas9-RNP. In conclusion, it elucidates that programmable Cas9-RNP is suitable for seamless gene editing in CC400. Moreover, the off-target effect can be decreased as well as the toxicity problem derived from long period expression of Cas9 protein can be solved due to Cas9-RNP degrade by itself which won't allow it to long-term stay in host cell.

[1] Apt, K. E., Grossman, A., and Kroth-Pancic, P. (1996). Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol.Gen.Genet. 252, 572-579.
[2] Bai, L. L., Yin, W. B., Chen, Y. H., Niu, L. L., Sun, Y. R., Zhao, S. M., et al. (2013). A new strategy to produce a defensin: stable production of mutated NP- 1 in nitratere ductase-deficient Chlorella ellipsoidea. PLoS ONE. 8:e54966.
[3] Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science. 315, 1709-1712.
[4] Baek, K., Kim, D. H., Jeong, J., Sim, S. J., Melis, A., Kim, J. S., Jin, E. S., & Bae, S., (2016). DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep. 6(1).
[5] Baek, K., Yu, J., Jeong, J., Sim, S. J., Bae, S., & Jin, E., (2017). Photoautotrophic production of macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA-free CRISPR-Cas9 RNP-mediated mutagenesis. Biotechnol. Bioeng. 115, 719-728.
[6] Bolotin, A., Quinquis, B., Sorokin, A., and Ehrlich, S.D. (2005). Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology.151, 2551-2561.
[7] Bruggeman, A. J., Kuehler, D., and Weeks, D. P., (2014). Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production. Plant. Biotechnol. J. 12, 894-902.
[8] Brouns, S. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J., Snijders, A. P., Dickman, M. J., Makarova, K. S., Koonin, E. V., and van der Oost, J. (2008). Small CRISPR RNAs g uide antiviral defense in prokaryotes. Science. 321, 960–964.
[9] Brown, L.E., Sprecher, S., and Keller, L., (1991). Introduction of exogenous DNA into Chlamydomonas reinhardtii by tobacco chloroplasts. J. Mol. Biol. 311, 1001-1009.
[10] Buzayan, J. M., Gerlach, W. L., Bruening, G., (1986). Satellite tobacco ringspot virus RNA: A subset of the RNA sequence is sufficient for autolytic processing. Proc. Natl. Acad. Sci. 3, 1131-1141.
[11] Carrier, T. A., Keasling, J. D., (1997). Engineering mRNA stability in E. coli by the addition of synthetic hairpins using a 5’ cassette system. Biotechnol Bioeng. 3, 577-580.
[12] Cerutti, H., Johnson, A. M., Gillham, N. W., and Boynton, J. E. (1997b). A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression. Genetics. 145, 97.
[13] Chen, Y., Wang, Y., Sun, Y., Zhang, L., & Li, W., (2001). Highly efficient expression of rabbit neutrophil peptide-1 gene in Chlorella ellipsoidea cells. Curr. Genet. 5-6, 365-370.
[14] Chavez, A., Scheiman, J., Vora, S., Pruitt, B. W., Tuttle M, Iyer, E., Lin, S., Kiani, S., Guzman, C. D., Wiegand, D. J., et al., (2015). Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 12, 326-8.
[15] Christou, P., (1993). Particle gun mediated transformation. Curr Opin Biotechnol. 2, 135-141.
[16] Clifton, K. P., Jones, E. M., Paudel, S., Marken, J. P., Monette, C. E., Halleran, A. D., Epp, L., and Saha, M. S., (2018). The genetic insulator RiboJ increases expression of insulated genes. J. Biol. Eng. 12, 23.
[17] Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., and Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823.
[18] Daboussi, F. et al., (2014). Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology. Nat Commun. 5:3831.
[19] Deveau, H., Barrangou, R., Garneau, J. E., Labonte´ , J., Fremaux, C., Boyaval, P., Romero, D. A., Horvath, P. and Moineau, S., (2008). Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390-1400.
[20] Deltcheva, E. et al., (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602-607.
[21] Desai, P. N., Shrivastava, N., Padh, H., (2010). Production of heterologous proteins in plants: Strategies for optimal expression. Biotechnol. Adv. 28, 427-435.
[22] Doron, L., Segal, N., and Shapira, M., (2016). Transgene expression in microalgae-from tools to applications. Front. Plant Sci. 7, 505.
[23] Dunahay, T., (1993). Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. Biotechniques. 15, 452-455, 457-458, 460.
[24] Endy, D., (2005). Foundations for engineering biology. Nature. 438, 449-453.
[25] El-Sheekh, M. M., (1999). Stable transformation of the intact cells of Chlorella kessleri with high velocity microprojectiles. Biologia Plantarum. 2, 209-216.
[26] Falciatore, A., Casotti, R., Leblanc, C., Abrescia, C., and Bowler, C., (1999). Transformation of nonselectable reporter genes in marine diatoms. Mar. Biotechnol. 1, 239-251.
[27] Ferenczi, A., Pyott, D. E., Xipnitou, A., and Molnar, A., (2017). Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA. PNAS. 114, 13567-13572.
[28] Fisher, A.L., Ohsako, S., and Caudy, M.,(1996). The WRPW motif of the hairyrelated basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain. Mol. Cell. Biol. 16, 2670-2677.
[29] Foster, A. J., Martin-Urdiroz, M., Yan, X., Wright, H. S., Soanes, D. M., Talbot N. J., (2018). CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus. Sci Rep. 8(1).
[30] Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R., (2017). Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 551, 464-471.
[31] Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V., (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. 109, 2579-2586.
[32] Garneau, J. E., Dupuis, M. E., Villion, M., Romero, D. A., Barrangou, R., Boyaval, P., Fremaux, C., Horvath, P., Magada´n, A. H., and Moineau, S. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67-71.
[33] Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A. et al. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 154, 442-451.
[34] Hall, L. M., Taylor, K. B., and Jones, D. D., (1993). Expression of a foreign gene in Chlamydomonas reinhardtii. Gene. 124, 75-81.
[35] Hawkins, R. L., and Nakamura, M., (1999). Expression of human growth hormone by the eukaryotic alga, Chlorella. Curr. Microbiol. 38, 335-341.
[36] Haft, D. H., Selengut, J., Mongodin, E. F., and Nelson, K. E. (2005). A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1, e60.
[37] Haag, A. L., (2007). Algae bloom again. Nature. 447, 520-521.
[38] Hathaway, N.A., Bell, O., Hodges, C., Miller, E.L., Neel, D.S., and Crabtree, G.R.,(2012). Dynamics and memory of heterochromatin in living cells. Cell.149, 1447-1460.
[39] Hopes, A., Nekrasov, V., Kamoun, S., Mock, T., (2016). Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. Plant Methods 12:49.
[40] Houdebine, L. M., (2009). Production of pharmaceutical proteins by transgenic animals. Comp. Immunol., Microbiol. Infect. Dis. 32, 107-121.
[41] Huang, C. H., Shen, C. R., Li, H., Sung, L. Y., Wu, M. Y., Hu, Y. C., (2016). CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S-elongatus PCC 7942. Microb Cell Fact. 15:196.
[42] Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., and Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169, 5429-5433.
[43] Jansen, R., Embden, J.D., Gaastra, W., and Schouls, L.M. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565-1575.
[44] Jarvis, E. E., and Brown, L. M., (1991). Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea. Curr.Genet. 19, 317-321.
[45] Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821.
[46] Jiang, W., Brueggeman, A. J., Horken, K. M., Plucinak, T. M., Weeks, D. P., (2014). Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii. Eukaryot Cell. 13, 1465-14969.
[47] Kao, P. H., Ng, I. S., (2017). CRISPRi mediated phosphoenolpyruvate carboxylase regulation to enhance the production of lipid in Chlamydomonas reinhardtii. Bioresour. Technol. 245,1527-1537.
[48] Khvorova, A., Lescoute, A., Westhof, E., and Jayasena, S. D., (2003). Sequencing elements outside the hammerhead ribozyme catalytic core enable intracellular activity. ‎Nat. Struct. Mol. Biol. 9, 708-712.
[49] Kim, S., Kim, D., Cho, S.W., Kim, J., Kim, J.S., (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012-9.
[50] Kim, D. H., Kim, Y. T., Cho, J. J., Bae, J. H. et al., (2002). Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea. Mar. Biotechnol. 4, 63-73.
[51] Kira, N., Ohnishi, K., Miyagawa-Yamaguchi, A., Kadono, T., and Adachi, M., (2015). Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment. Mar. Genomics. 25, 49-56.
[52] Kindle, K. L., Schnell, R. A., Fernandez, E., and Lefebvre, P. A., (1989). Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J. CellBiol. 109, 2589-2601.
[53] Kindle, K. L., (1990). High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. 87, 1228-1232.
[54] Kilian, O., Benemann, C. S., Niyogi, K. K., Vick, B., (2011). High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proc. Natl. Acad. Sci. 108, 21265-21269.
[55] Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Topkar, V. V., Zheng, Z., and Joung, J. K., (2015). Broadening Staphylococcus aureus Cas9 targeting range by modifying PAM recognition. 33,1293-1298.
[56] Kovar, J. L., Zhang, J., Funke, R. P., and Weeks, D. P. (2002). Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation. PlantJ. 29, 109-117.
[57] Kumar, S. V., Misquitta, R. W., Reddy, V. S., Rao, B. J., Rajam, M. V., (2004). Genetic transformation of the green alga—Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Science. 166, 731-738.
[58] La Russa, M. F., Qi, L. S., (2015). The new state of the art: Cas9 for gene activation and repression. Mol. Cell. Biol. 35, 3800-9.
[59] Lapidot, M., Raveh, D., Sivan, A., Arad, S. M., and Shapira, M. (2002). Stable chloroplast transformation of the unicellular red alga Porphyridium Sp. Plant Physiol. 129, 7-12.
[60] León-Bañares, R., (2004). Transgenic microalgae as green cell-factories. Trends Biotechnol. 22, 45-52.
[61] Lin, H. D., Liu, B. H., Kuo, T. T., Tsai, H. C. et al., (2013). Knockdown of PsbO leads to induction of HydA and production of photobiological H2 in the green alga Chlorella sp. DT. Bioresour. Technol. 143, 154-162.
[62] Lin, H., Kwan, A. L., and Dutcher, S. K., (2010). Synthesizing and salvaging NAD: lessons learned from Chlamydomonas reinhardtii. PLoSGenet. 6:e1001105.
[63] Lin, S., Staahl, B. T., Alla, R. L., Doudna, J. A., (2014). Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 3.
[64] Lin, S., Qiao,J., Ma, L., Liu, Y., (2018). Direct purification of CRISPR/Cas ribonucleoprotein from E. coli in a single step. bioRxiv.
[65] Liu, J., Sun, Z., Gerken, H., Huang, J., Jiang, Y., and Chen, F., (2014). Genetic engineering of the green alga Chlorella zofingiensis: a modified norflurazon- resistant phytoene desaturase gene as a dominant selectable marker. Appl. Microbiol. Biotechnol. 98, 5069-5079.
[66] Liu, L., Wang, Y., Zhang, Y., Chen, X. et al., (2013). Development of a new method for genetic transformation of the green alga Chlorella ellipsoidea. Mol. Biotechnol. 54, 211-219.
[67] Li, H., Shen, C. R., Huang, C. H., Sung, L. Y., Wu, M. Y., Hu, Y. C., (2016). CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production. Metab Eng. 38, 293-302.
[68] Li, J., Xue, L., Yan, H., Liu, H., and Liang, J., (2008). Inducible EGFP expression under the control of the nitrate reductase gene promoter in transgenic Dunaliella salina. J. Appl. Phycol. 20, 137-145.
[69] Li, W., Wang, F., Qiao, J., Liu, Y., and Ma, L., (2018). Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly. bioRxiv.
[70] Lohuis, M. R., and Miller, D. J., (1998). Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. Plant J. 13, 427-435.
[71] Lou, C., Stanton, B., Chen, Y. J., Munsky, B,. and Voigt, C. A., (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 11, 1137-1142.
[72] Mayfield, S. P., and Kindle, K. L., (1990). Stable nuclear transformation of Chlamydomonas reinhardtii by using a C.reinhardtii gene as the selectable marker. Proc. Natl. Acad. Sci. 87, 2087-2091.
[73] Makarova, K.S., Grishin, N.V., Shabalina, S.A., Wolf, Y.I. and Koonin, E.V. (2006). A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct. 1, 7.
[74] Margolin, J.F. et al. (1994). Krüppel-associated boxesarepotent transcriptional repression domains. Proc. Natl. Acad. Sci. 91, 4509-4513.
[75] Molnar, A., Bassett, A., Thuenemann, E., Schwach, F., Karkare, S., Ossowski, S., et al. (2009). Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. PlantJ. 58,165-174.
[76] Mojica, F.J., Díez‐Villaseñor, C., Soria, E., and Juez, G. (2000). Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol. Microbiol. 36, 244-246.
[77] Mojica, F. J., Díez-Villaseñor, C., García-Martínez, J., Soria, E. (2005). Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 2, 174-182.
[78] Mojica, F. J., Díez‐Villaseñor, C., García-Martínez, J. and Almendros, C., (2009). Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology. 3, 733-740.
[79] Nelson, J., Savereide, P. B., and Lefebvre, P. A., (1994). The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation. Mol.Cell.Biol. 14, 4011-4019.
[80] Nelson, J. A., Shepotinovskaya, I., and Uhlenbeck, O. C., (2005), Hammerheads derived from sTRSV show enhanced cleavage and ligation rate constants. Biochemistry. 44, 14577-14585.
[81] Neupert, J., Karcher, D. and Bock, R., (2009). Generation of Chlamydomonas strains that efficiently express nuclear transgenes. PlantJ. 57, 1140-1150.
[82] Nymark, M., Sharma, A. K., Sparstad, T., Bones, A. M., Winge, P., (2016). A CRISPR/Cas9 system adapted for genome editing in marine algae. Sci Rep. 6:24951.
[83] Pauwels, K., Podevin, N., Breyer, D., Carroll, D., Herman, P.. (2014). Engineering nucleases for gene targeting: safety and regulatory considerations. New Biotechnol. 31, 18-27.
[84] Perez-Pinera, P. et al. (2013) RNA-guided gene activation by CRISPR–Cas9-based transcription factors. Nat. Methods. 10, 97-976.
[85] Pourcel, C., Salvignol, G.,Vergnaud, G. (2005). CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 3, 653-663.
[86] Poliner, E., Takeuchi, T., Du, Z. Y., Benning, C., Farre. E. M., (2018) Nontransgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779. ACS Synth. Biol. 7, 962-968.
[87] Porro, D., Gasser, B., Fossati, T., Maurer, M. et al., (2011). Production of recombinant proteins and metabolites in yeasts. Appl. Microbiol. Biotechnol. 89, 939-948.
[88] Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A., (2013). Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 152, 1173-1183.
[89] Qi, L., Haurwitz, R. E., Shao, W., Doudna, J. A., Arkin, A. P., (2012). RNA processing enables predictable programming of gene expression. Nat. Biotechnol. 10, 1002-1006.
[90] Radakovits, R., Eduafo, P. M., Posewitz, M. C., (2011). Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum. Metab. Eng. 13, 89-95.
[91] Rajagopalan, N., Kagale, S., Bhowmik, P. and Song, H., (2018). A two-step method for obtaining highly pure Cas9 nuclease for genome editing, biophysical, and structural studies. Methods and Protoc. 1, 17.
[92] Rochaix, J. D., & van Dillewijn, J. (1982). Transformation of the green alga Chlamydomonas reinhardii with yeast DNA. Nature. 296, 70-72.
[93] San Cha, T., Yee, W., Aziz, A., (2012). Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris. World J. Microbiol. Biotechnol. 28, 1771-1779.
[94] Schroda, M. et al. (2000). The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J. 21, 121-131.
[95] Schmollinger, S., Strenkert, D., and Schroda, M., (2010). An inducible artificial microRNA system for Chlamydomonas reinhardtii confirms a key role for heat shock factor 1 in regulating thermotolerance. Curr. Genet. 56, 383-389.
[96] Sharon-Gojman, R., Maimon, E., Leu, S., Zarka, A., and Boussiba, S., (2015). Advanced methods for genetic engineering of Haematococcus pluvialis (Chlorophyceae, Volvocales). AlgalRes. 10,8-15.
[97] Shin et al. (2016). CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii. Sci Rep. 6, 27810.
[98] Shimizu, Y., (1996). Microalgal metabolites: a new perspective. Annu. Rev. Microbiol. 50, 431-465.
[99] Shmakov, S., Abudayyeh, O. O., Makarova, K. S., Wolf, Y. I., Gootenberg, J. S., Semenova, E., Koonin, E. V., (2015). Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Molecular Cell. 60, 385-397.
[100] Sizova, I. et al. (2001). A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene. 277, 221-229.
[101] Sizova, I., Greiner, A., Awasthi, M., Kateriya, S., Hegemann, P., (2013).Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases. Plant J. 73, 873-882.
[102] Skjånes, K., Rebours, C., & Lindblad, P., (2013). Potential for green microalgae to produce hydrogen, pharmaceuticals and other high value products in a combined process. Crit. Rev. Biotechnol. 33, 172-215.
[103] Staahl, B. T., Benekareddy, M., Coulon-Bainier, C., Banfal, A. A., Floor, S. N., Sabo, J. K., Urnes, C., Munares, G. A., Ghosh, A., Doudna, J. A., (2017). Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 5, 431-434.
[104] Stevens, D. R., Purton, S., and Rochaix, J. D., (1996).The bacterial phleomycin resistance geneble as a dominant selectable marker in Chlamydomonas. Mol. Gen.Genet. 251,23-30.
[105] Sun, G., Zhang, X., Sui, Z., and Mao, Y., (2008). Inhibition of pds gene expression via the RNA interference approach in Dunaliella salina (Chlorophyta). Mar. Biotechnol. 10, 219-226.
[106] Sung, Y. H. et al. (2014). Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 24, 125-131.
[107] Svitashev, S., Schwartz, C., Lenderts, B., Young, J. K., & Cigan, A. M., (2016). Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun. 16, 7:13274.
[108] Tang, D. K. H. et al., (1995). Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA. Biochem. Mol.Biol. Int. 36, 1025-1035.
[109] Tanenbaum, M. E., Gilbert, L.A., Qi, L.S., Weissman, J.S., Vale, R. D., (2014). A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 3, 635-646.
[110] Wang, C., Wang, Y., Su, Q., Gao, X., (2007). Transient expression of the GUS gene in a unicellular marine green alga, Chlorella sp. MACC/C95, via electroporation. Biotechnol. Bioprocess Eng. 12, 180-183.
[111] Wang, Q., Lu, Y., Xin, Y., Wei, L., Huang, S., Xu, J., (2016). Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9. Plant J. 6, 1071-1081.
[112] Wang, P., Sun, Y., Li, X., Zhang, L., Li, W., and Wang, Y., (2004). Rapid isolation and functional analysis of promoter sequences of the nitrate reductase gene from Chlorella ellipsoidea. J. Appl. Phycol. 16, 11-16.
[113] Wang, L., Yang, L., Wen, X., Chen, Z., Liang, Q., Li, J., and Wang, W., (2019). Rapid and high efficiency transformation of Chlamydomonas reinhardtii by square-wave electroporation. Bioscience. Rep. 39, 1.
[114] Wendt, K. E., Ungerer, J., Cobb, R.E., Zhao, H.M., Pakrasi, H.B., (2016). CRISPR/ Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. Microb. Cell. Fact. 15(1).
[115] Woo, K. W., Kim, J., Kwon, S. I., Corvalán, C., Cho, S. W., Kim, H., Kim, S. G., Kim, S. T., Choe, S., & Kim, J. S., (2015). DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol. 33, 1162-4.
[116] Yao, L., Cengic, I., Anfelt, J., Hudson, E. P., (2016). Multiple gene repression in Cyanobacteria using CRISPRi. ACS Synth. Biol. 5, 207-212.
[117] Yin, H., Xue, W., Chen, S., Bogorad, R. L., Benedetti, E., Grompe, M., Koteliansky, V., Sharp, P. A., Jacks, T., Anderson, D. G., (2014). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol. 32, 551-553.
[118] Yoshimitsu, Y., Abe, J., and Harayama, S., (2018). Cas9‑guide RNA ribonucleoprotein‑induced genome editing in the industrial green alga Coccomyxa sp. strain KJ. Biotechnol. Biofuels. 11(1).
[119] Zaslavskaia, L. A., Lippmeier, J. C., Kroth, P. G., Grossman, A. R., and Apt, K. E., (2000). Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. J. Phycol. 36, 379-386.
[120] Zetsche, B.et al., (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 163, 759-771.