Gene Editing Technology of CRISPR/Cas and Its Applications in Microalgae Research

doi:10.13560/j.cnki.biotech.bull.1985.2021-0877

Abstract

Abstract:

Due to their potentials in medicine,food,renewable fuels and chemical raw materials,microalgae has attracted more and more attentions from current researchers. However,the progress of microalgae in genetic engineering is relatively slow because it is lack of suitable gene editing methods and transformation tools. With the development of molecular biology and gene editing technology,CRISPR technology has emerged as a powerful method for studying gene function,improving plant breeding and increasing metabolite products with its advantages of simplicity,specificity and efficiency. Based on these,we introduced the two main types of CRISPR/Cas,focusing on the application progress of CRISPR in microalgae,and summarized the issues existing in the application of CRISPR technology in microalgae,aiming to provide inspiration and reference for future research.

Key words: gene editing, CRISPR/Cas, microalgae, Cas9, transformation

CHEN Ying-dan, ZHANG Yang, XIA Qiang, SUN Hong-xia. Gene Editing Technology of CRISPR/Cas and Its Applications in Microalgae Research[J]. Biotechnology Bulletin, 2022, 38(5): 257-268.

Figures/Tables 3

References 79

[1]	Singh A, Nigam PS, Murphy JD. Mechanism and challenges in commercialisation of algal biofuels[J]. Bioresour Technol, 2011, 102(1):26-34. doi: 10.1016/j.biortech.2010.06.057 URL
[2]	Rizwan M, Mujtaba G, Memon SA, et al. Exploring the potential of microalgae for new biotechnology applications and beyond:a review[J]. Renew Sustain Energy Rev, 2018, 92:394-404. doi: 10.1016/j.rser.2018.04.034 URL
[3]	Sharma K, Dhruv S, Mani U, et al. Therapeutic utility of Spirulina[M]// Spirulina in Human Nutrition and Health. Boca Raton: CRC Press, 2007:71-99.
[4]	Yaakob Z, Ali E, Zainal A, et al. An overview:biomolecules from microalgae for animal feed and aquaculture[J]. J Biol Res:Thessalon, 2014, 21(1):6. doi: 10.1186/2241-5793-21-6 URL
[5]	Spolaore P, Joannis-Cassan C, Duran E, et al. Commercial applications of microalgae[J]. J Biosci Bioeng, 2006, 101(2):87-96. pmid: 16569602
[6]	Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications:a review[J]. Renew Sustain Energy Rev, 2010, 14(1):217-232. doi: 10.1016/j.rser.2009.07.020 URL
[7]	Abdel-Raouf N. Agricultural importance of algae[J]. Afr J Biotechnol, 2012, 11(54):11648-11658.
[8]	Renuka N, Guldhe A, Prasanna R, et al. Microalgae as multi-functional options in modern agriculture:current trends, prospects and challenges[J]. Biotechnol Adv, 2018, 36(4):1255-1273. doi: 10.1016/j.biotechadv.2018.04.004 URL
[9]	Muñoz R, Guieysse B. Algal-bacterial processes for the treatment of hazardous contaminants:a review[J]. Water Res, 2006, 40(15):2799-2815. pmid: 16889814
[10]	Wilde EW, Benemann JR. Bioremoval of heavy metals by the use of microalgae[J]. Biotechnol Adv, 1993, 11(4):781-812. pmid: 14538057
[11]	Ishino Y, Shinagawa H, Makino K, et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[J]. J Bacteriol, 1987, 169(12):5429-5433. doi: 10.1128/jb.169.12.5429-5433.1987 pmid: 3316184
[12]	Hermans PW, van Soolingen D, Bik EM, et al. Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains[J]. Infect Immun, 1991, 59(8):2695-2705. doi: 10.1128/iai.59.8.2695-2705.1991 pmid: 1649798
[13]	Mojica FJ, Juez G, Rodríguez-Valera F. Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites[J]. Mol Microbiol, 1993, 9(3):613-621. pmid: 8412707
[14]	Mojica FJ, Ferrer C, Juez G, et al. Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning[J]. Mol Microbiol, 1995, 17(1):85-93. pmid: 7476211
[15]	Bult CJ, White O, Olsen GJ, et al. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii[J]. Science, 1996, 273(5278):1058-1073. pmid: 8688087
[16]	Nelson KE, Clayton RA, Gill SR, et al. Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima[J]. Nature, 1999, 399(6734):323-329. doi: 10.1038/20601 URL
[17]	Mojica FJ, Díez-Villaseñor C, Soria E, et al. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria[J]. Mol Microbiol, 2000, 36(1):244-246. pmid: 10760181
[18]	Jansen R, Embden JD, Gaastra W, et al. Identification of genes that are associated with DNA repeats in prokaryotes[J]. Mol Microbiol, 2002, 43(6):1565-1575. pmid: 11952905
[19]	Mojica FJ, Díez-Villaseñor C, García-Martínez J, et al. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[J]. J Mol Evol, 2005, 60(2):174-182. doi: 10.1007/s00239-004-0046-3 URL
[20]	Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes[J]. Science, 2007, 315(5819):1709-1712. doi: 10.1126/science.1138140 pmid: 17379808
[21]	Brouns SJ, Jore MM, Lundgren M, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes[J]. Science, 2008, 321(5891):960-964. doi: 10.1126/science.1159689 pmid: 18703739
[22]	Garneau JE, Dupuis MÈ, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA[J]. Nature, 2010, 468(7320):67-71. doi: 10.1038/nature09523 URL
[23]	Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J]. PNAS, 2012, 109(39):E2579-E2586.
[24]	Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096):816-821. doi: 10.1126/science.1225829 URL
[25]	Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121):819-823. doi: 10.1126/science.1231143 pmid: 23287718
[26]	Makarova KS, Wolf YI, Iranzo J, et al. Evolutionary classification of CRISPR-Cas systems:a burst of class 2 and derived variants[J]. Nat Rev Microbiol, 2020, 18(2):67-83. doi: 10.1038/s41579-019-0299-x pmid: 31857715
[27]	Anders C, Niewoehner O, Duerst A, et al. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease[J]. Nature, 2014, 513(7519):569-573. doi: 10.1038/nature13579 URL
[28]	Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system[J]. Cell, 2015, 163(3):759-771. doi: 10.1016/j.cell.2015.09.038 pmid: 26422227
[29]	Swarts DC, Jinek M. Cas9 versus Cas12a/Cpf1:Structure-function comparisons and implications for genome editing[J]. Wiley Interdiscip Rev RNA, 2018:e1481.
[30]	Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression[J]. Cell, 2013, 152(5):1173-1183. doi: 10.1016/j.cell.2013.02.022 URL
[31]	Cleto S, Jensen JV, Wendisch VF, et al. Corynebacterium glutamicum metabolic engineering with CRISPR interference(CRISPRi)[J]. ACS Synth Biol, 2016, 5(5):375-385. doi: 10.1021/acssynbio.5b00216 pmid: 26829286
[32]	Feng Z, Zhang B, Ding W, et al. Efficient genome editing in plants using a CRISPR/Cas system[J]. Cell Res, 2013, 23(10):1229-1232. doi: 10.1038/cr.2013.114 URL
[33]	Jiang W, Zhou H, Bi H, et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, Sorghum and rice[J]. Nucleic Acids Res, 2013, 41(20):e188. doi: 10.1093/nar/gkt780 URL
[34]	Upadhyay SK, Kumar J, Alok A, et al. RNA-guided genome editing for target gene mutations in wheat[J]. G3:Bethesda, 2013, 3(12):2233-2238.
[35]	Jacobs TB, LaFayette PR, Schmitz RJ, et al. Targeted genome modifications in soybean with CRISPR/Cas9[J]. BMC Biotechnol, 2015, 15:16. doi: 10.1186/s12896-015-0131-2 URL
[36]	杨柳, 李晓峰, 祝万万, 等. 利用CRISPR-Cas9基因编辑技术获得水稻OsMADS56基因突变体[J]. 分子植物育种, 2020, 18(11):3571-3578.
	Yang L, Li XF, Zhu WW, et al. Generation of OsMADS56 mutants in rice using CRISPR/Cas9 editing approach[J]. Mol Plant Breed, 2020, 18(11):3571-3578.
[37]	林萌萌, 李春娟, 闫彩霞, 等. CRISPR/Cas9基因编辑技术在作物中的应用[J]. 核农学报, 2021, 35(6):1329-1339.
	Lin MM, Li CJ, Yan CX, et al. Application of CRISPR/Cas9 gene editing technology in crops[J]. J Nucl Agric Sci, 2021, 35(6):1329-1339.
[38]	涂文凤, 王月, 杨文强. 基因编辑技术在莱茵衣藻中的应用进展[J]. 生命科学, 2018, 30(9):987-993.
	Tu WF, Wang Y, Yang WQ. Progress in the application of genome editing in Chlamydomonas reinhardtii[J]. Chin Bull Life Sci, 2018, 30(9):987-993.
[39]	Merchant SS, Prochnik SE, Vallon O, et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions[J]. Science, 2007, 318(5848):245-250. doi: 10.1126/science.1143609 pmid: 17932292
[40]	Arriola MB, Velmurugan N, Zhang Y, et al. Genome sequences of Chlorella sorokiniana UTEX 1602 and Micractinium conductrix SAG 241. 80:implications to maltose excretion by a green alga[J]. Plant J, 2018, 93(3):566-586. doi: 10.1111/tpj.13789 URL
[41]	Kindle KL, Schnell RA, Fernández E, et al. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase[J]. J Cell Biol, 1989, 109(6 pt 1):2589-2601. pmid: 2592399
[42]	Mussgnug JH. Genetic tools and techniques for Chlamydomonas reinhardtii[J]. Appl Microbiol Biotechnol, 2015, 99(13):5407-5418. doi: 10.1007/s00253-015-6698-7 pmid: 26025017
[43]	Jiang W, Brueggeman AJ, Horken KM, et al. Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii[J]. Eukaryot Cell, 2014, 13(11):1465-1469. doi: 10.1128/EC.00213-14 URL
[44]	Shin SE, Lim JM, Koh HG, et al. CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii[J]. Sci Rep, 2016, 6:27810. doi: 10.1038/srep27810 URL
[45]	Baek K, Kim DH, Jeong J, et al. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins[J]. Sci Rep, 2016, 6:30620. doi: 10.1038/srep30620 URL
[46]	Baek K, Yu J, Jeong J, et al. Photoautotrophic production of macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA-free CRISPR-Cas9 RNP-mediated mutagenesis[J]. Biotechnol Bioeng, 2018, 115(3):719-728. doi: 10.1002/bit.26499 URL
[47]	Kao PH, Ng IS. CRISPRi mediated phosphoenolpyruvate carboxylase regulation to enhance the production of lipid in Chlamydomonas reinhardtii[J]. Bioresour Technol, 2017, 245(pt b):1527-1537. doi: 10.1016/j.biortech.2017.04.111 URL
[48]	Dhokane D, Bhadra B, Dasgupta S. CRISPR based targeted genome editing of Chlamydomonas reinhardtii using programmed Cas9-gRNA ribonucleoprotein[J]. Mol Biol Rep, 2020, 47(11):8747-8755. doi: 10.1007/s11033-020-05922-5 URL
[49]	Bižić M, Klintzsch T, Ionescu D, et al. Aquatic and terrestrial cyanobacteria produce methane[J]. Sci Adv, 2020, 6(3):eaax5343. doi: 10.1126/sciadv.aax5343 URL
[50]	Wang L, Chen L, Yang S, et al. Photosynthetic conversion of carbon dioxide to oleochemicals by cyanobacteria:recent advances and future perspectives[J]. Front Microbiol, 2020, 11:634. doi: 10.3389/fmicb.2020.00634 pmid: 32362881
[51]	Wendt KE, Ungerer J, Cobb RE, et al. CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973[J]. Microb Cell Fact, 2016, 15(1):115. doi: 10.1186/s12934-016-0514-7 URL
[52]	Li H, Shen CR, Huang CH, et al. CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production[J]. Metab Eng, 2016, 38:293-302. doi: 10.1016/j.ymben.2016.09.006 URL
[53]	Ungerer J, Pakrasi HB. Cpf1 is A versatile tool for CRISPR genome editing across diverse species of cyanobacteria[J]. Sci Rep, 2016, 6:39681. doi: 10.1038/srep39681 pmid: 28000776
[54]	Yao L, Cengic I, Anfelt J, et al. Multiple gene repression in cyanobacteria using CRISPRi[J]. ACS Synth Biol, 2016, 5(3):207-212. doi: 10.1021/acssynbio.5b00264 URL
[55]	Huang CH, Shen CR, Li H, et al. CRISPR interference(CRISPRi)for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942[J]. Microb Cell Fact, 2016, 15(1):196. doi: 10.1186/s12934-016-0595-3 URL
[56]	Higo A, Isu A, Fukaya Y, et al. Application of CRISPR interference for metabolic engineering of the heterocyst-forming multicellular cyanobacterium Anabaena sp. PCC 7120[J]. Plant Cell Physiol, 2018, 59(1):119-127. doi: 10.1093/pcp/pcx166 URL
[57]	Cho S, Choe D, Lee E, et al. High-level dCas9 expression induces abnormal cell morphology in Escherichia coli[J]. ACS Synth Biol, 2018, 7(4):1085-1094. doi: 10.1021/acssynbio.7b00462 URL
[58]	Choi SY, Woo HM. CRISPRi-dCas12a:a dCas12a-mediated CRISPR interference for repression of multiple genes and metabolic engineering in cyanobacteria[J]. ACS Synth Biol, 2020, 9(9):2351-2361. doi: 10.1021/acssynbio.0c00091 URL
[59]	Kaczmarzyk D, Cengic I, Yao L, et al. Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX[J]. Metab Eng, 2018, 45:59-66. doi: S1096-7176(17)30383-X pmid: 29199103
[60]	Nymark M, Sharma AK, Sparstad T, et al. A CRISPR/Cas9 system adapted for gene editing in marine algae[J]. Sci Rep, 2016, 6:24951. doi: 10.1038/srep24951 URL
[61]	Hopes A, Nekrasov V, Kamoun S, et al. Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana[J]. Plant Methods, 2016, 12:49. doi: 10.1186/s13007-016-0148-0 URL
[62]	Stukenberg D, Zauner S, Dell’Aquila G, et al. Optimizing CRISPR/Cas9 for the diatom Phaeodactylum tricornutum[J]. Front Plant Sci, 2018, 9:740. doi: 10.3389/fpls.2018.00740 pmid: 29928285
[63]	Nawaly H, Tsuji Y, Matsuda Y. Rapid and precise genome editing in a marine diatom, Thalassiosira pseudonana by Cas9 nickase(D10A)[J]. Algal Res, 2020, 47:101855. doi: 10.1016/j.algal.2020.101855 URL
[64]	Moosburner MA, Gholami P, McCarthy JK, et al. Multiplexed knockouts in the model diatom Phaeodactylum by episomal delivery of a selectable Cas9[J]. Front Microbiol, 2020, 11:5. doi: 10.3389/fmicb.2020.00005 pmid: 32047486
[65]	Wang Q, Lu Y, Xin Y, et al. Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9[J]. Plant J, 2016, 88(6):1071-1081. doi: 10.1111/tpj.13307 URL
[66]	Ajjawi I, Verruto J, Aqui M, et al. Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator[J]. Nat Biotechnol, 2017, 35(7):647-652. doi: 10.1038/nbt.3865 pmid: 28628130
[67]	Lin WR, Ng IS. Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation[J]. Enzyme Microb Technol, 2020, 133:109458. doi: 10.1016/j.enzmictec.2019.109458 URL
[68]	Altpeter F, Springer NM, Bartley LE, et al. Advancing crop transformation in the era of genome editing[J]. Plant Cell, 2016, 28(7):1510-1520.
[69]	Boynton JE, Gillham NW, Harris EH, et al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles[J]. Science, 1988, 240(4858):1534-1538. pmid: 2897716
[70]	Kindle KL. High-frequency nuclear transformation of Chlamydomonas reinhardtii[J]. PNAS, 1990, 87(3):1228-1232. pmid: 2105499
[71]	Dunahay TG, Adler SA, Jarvik JW. Transformation of microalgae using silicon carbide whiskers[M]// Rocky S. Tuan. Recombinant gene expression protocols, Totowa: Humana Press, 1997:pp 503-509.
[72]	Yamano T, Iguchi H, Fukuzawa H. Rapid transformation of Chlamydomonas reinhardtii without cell-wall removal[J]. J Biosci Bioeng, 2013, 115(6):691-694. doi: 10.1016/j.jbiosc.2012.12.020 URL
[73]	Fabris M, George J, Kuzhiumparambil U, et al. Extrachromosomal genetic engineering of the marine diatom Phaeodactylum tricornutum enables the heterologous production of monoterpenoids[J]. ACS Synth Biol, 2020, 9(3):598-612. doi: 10.1021/acssynbio.9b00455 URL
[74]	Porter RD. Transformation in cyanobacteria[J]. Crit Rev Microbiol, 1986, 13(2):111-132. pmid: 3095029
[75]	Lanigan TM, Kopera HC, Saunders TL. Principles of genetic engineering[J]. Genes, 2020, 11(3):291. doi: 10.3390/genes11030291 URL
[76]	Slattery SS, Diamond A, Wang H, et al. An expanded plasmid-based genetic toolbox enables Cas9 genome editing and stable maintenance of synthetic pathways in Phaeodactylum tricornutum[J]. ACS Synth Biol, 2018, 7(2):328-338. doi: 10.1021/acssynbio.7b00191 pmid: 29298053
[77]	Kim S, Kim D, Cho SW, et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins[J]. Genome Res, 2014, 24(6):1012-1019. doi: 10.1101/gr.171322.113 URL
[78]	Wu-Scharf D, Jeong B, Zhang C, et al. Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase[J]. Science, 2000, 290(5494):1159-1162. pmid: 11073454
[79]	姜涛. 基因编辑之刑法规制及其限度[J]. 东方法学, 2021(2):69-85.
	Jiang T. Criminal regulation of gene editing and its limits[J]. Orient Law, 2021(2):69-85.

特征Characteristic	Cas9	Cpf1
CRISPR分类	Class 2,type II Class 2,type V
来源	Streptococcus pyogenes	Francisella novicida Acidaminococcussp. Lachnospiraceae bacterium Moraxella bovoculi
核酸酶结构域	HNH、RuvC	RuvC
CRISPR-RNA	crRNA、tracrRNA	crRNA
PAM	NGG	TTTN
切割特点	平末端	黏性末端
RNAase活性	无	有

特征Characteristic	Cas9	Cpf1
CRISPR分类	Class 2,type II Class 2,type V
来源	Streptococcus pyogenes	Francisella novicida Acidaminococcussp. Lachnospiraceae bacterium Moraxella bovoculi
核酸酶结构域	HNH、RuvC	RuvC
CRISPR-RNA	crRNA、tracrRNA	crRNA
PAM	NGG	TTTN
切割特点	平末端	黏性末端
RNAase活性	无	有

类别 Category	微藻类型 Types of microalgae	方法 Method	目标基因 Target gene	相关内容 Related information	参考文献 Reference
衣藻	Chlamydonomas reinhardtii CC503	CRISPR/Cas9	FKB12	CRISPR/Cas9系统首次应用在微藻	[40]
	Chlamydonomas reinhardtii CC-124	CRISPR/Cas9、RNP	MAA7、CpSRP43、ChlM	靶向敲除或敲入,并通过NHEJ修复	[41]
	Chlamydonomas reinhardtii CC-4349	CRISPR/Cas9、RNP	ZEP、CpFTSY	实现了双基因敲除,产生突变体	[42]
	Chlamydonomas reinhardtii CC-4349	CRISPR/Cas9、RNP	ZEP	ZEP敲除突变体能够产生大量的叶黄素和玉米黄质	[43]
	Chlamydonomas reinhardtii CC-2931 Chlamydonomas reinhardtii CC-1883	CRISPR / Cpf1、RNP	FKB12、CpFTSY、CpSRP43、PHT7	Cpf1核酸酶首次应用于莱茵衣藻	[44]
	Chlamydonomas reinhardtii CC-400	CRISPR/dCas9	PEPC1、RFP	CRISPRi系统首次应用在莱茵衣藻	[45]
	Chlamydonomas reinhardtii CC-125	CRISPR/Cas9、RNP	MAA7	优化CRISPR RNP流程,提高基因编辑效率	[46]
蓝藻	Synechococcus elongatus UTEX 2973	CRISPR/Cas9	nblA	这是在蓝藻中使用CRISPR / Cas9基因组编辑的第一份报告	[49]
	Synechococcus elongatus PCC 7942	CRISPR/Cas9	glgc	glgC缺失突变体产生更高水平的琥珀酸酯	[50]
	Synechococcus UTEX 2973	CRISPR/Cpf1	psbA1、nblA	比较Cpf1和Cas9的毒性;Cpf1是蓝藻基因组编辑的合适核酸酶	[51]
	Synechocystis 6803 Anabaena 7120	CRISPR/Cpf1	nblA、nifH	缺失突变、点突变以及插入突变,研究了Cpf1的多功能性	[51]
	Synechcocystis sp. PCC 6803	CRISPR/dCas9	phaE、glgC	抑制碳存储化合物聚羟基丁酸酯（PHB）和糖原的形成。	[52]
	Synechococcus elongatus PCC 7942	CRISPR/dCas9	glgc、sdhA、sdhB	增加了琥珀酸产量	[53]
	Anabaena sp. PCC 7120	CRISPR/dCas9	glnA	成功微调glnA的表达水平;控制铵的生产。	[54]
	Synechocystis sp. PCC 6803	CRISPR/dCas9	PlsX	抑制必需的酰基转移酶PlsX可将脂肪醇滴度提高3倍。	[57]
	Synechococcus elongatus PCC 7942	CRISPR/dCpf1	nblA、acnB、cpcB2	使用CRISPR-dCas12a改善光合角鲨烯的生产	[56]
硅藻	Phaeodactylum tricornutum	CRISPR/Cas9	CpSRP54	CRISPR/Cas9首次在硅藻的应用	[58]
	Thalassiosira pseudonana	CRISPR/Cas9	urease	脲酶基因的精确删除	[59]
	Phaeodactylum tricornutum	CRISPR/Cas9	vtc2、pho4	优化方法,产生单等位基因突变和双等位基因突变	[60]
	Thalassiosira pseudonana	CRISPR/Cas9	TpθCA3	使用Cas9切口酶介导的基因组编辑,获得了海洋硅藻突变体	[61]
	Phaeodactylum tricornutum	CRISPR/Cas9	NR、GS-2、cGOGAT	新的Cas9附加体设计减少了生产和筛选突变菌株的时间	[62]
其他	Nannochloropsis oceanica IMET1	CRISPR/Cas9	Nitrate reductase gene、HygR	含油微藻的基因编辑,突变菌株敲除效率提高	[63]
	Nannochloropsis gaditana	CRISPR/Cas9	ZnCys	微调ZnCys表达,优化脂质生产	[64]
	Chlorella vulgarisFSP-E	CRISPR/Cas9	fad3	CRISPR / Cas9系统是首次应用于小球藻FSP-E	[65]

类别 Category	微藻类型 Types of microalgae	方法 Method	目标基因 Target gene	相关内容 Related information	参考文献 Reference
衣藻	Chlamydonomas reinhardtii CC503	CRISPR/Cas9	FKB12	CRISPR/Cas9系统首次应用在微藻	[40]
	Chlamydonomas reinhardtii CC-124	CRISPR/Cas9、RNP	MAA7、CpSRP43、ChlM	靶向敲除或敲入,并通过NHEJ修复	[41]
	Chlamydonomas reinhardtii CC-4349	CRISPR/Cas9、RNP	ZEP、CpFTSY	实现了双基因敲除,产生突变体	[42]
	Chlamydonomas reinhardtii CC-4349	CRISPR/Cas9、RNP	ZEP	ZEP敲除突变体能够产生大量的叶黄素和玉米黄质	[43]
	Chlamydonomas reinhardtii CC-2931 Chlamydonomas reinhardtii CC-1883	CRISPR / Cpf1、RNP	FKB12、CpFTSY、CpSRP43、PHT7	Cpf1核酸酶首次应用于莱茵衣藻	[44]
	Chlamydonomas reinhardtii CC-400	CRISPR/dCas9	PEPC1、RFP	CRISPRi系统首次应用在莱茵衣藻	[45]
	Chlamydonomas reinhardtii CC-125	CRISPR/Cas9、RNP	MAA7	优化CRISPR RNP流程,提高基因编辑效率	[46]
蓝藻	Synechococcus elongatus UTEX 2973	CRISPR/Cas9	nblA	这是在蓝藻中使用CRISPR / Cas9基因组编辑的第一份报告	[49]
	Synechococcus elongatus PCC 7942	CRISPR/Cas9	glgc	glgC缺失突变体产生更高水平的琥珀酸酯	[50]
	Synechococcus UTEX 2973	CRISPR/Cpf1	psbA1、nblA	比较Cpf1和Cas9的毒性;Cpf1是蓝藻基因组编辑的合适核酸酶	[51]
	Synechocystis 6803 Anabaena 7120	CRISPR/Cpf1	nblA、nifH	缺失突变、点突变以及插入突变,研究了Cpf1的多功能性	[51]
	Synechcocystis sp. PCC 6803	CRISPR/dCas9	phaE、glgC	抑制碳存储化合物聚羟基丁酸酯（PHB）和糖原的形成。	[52]
	Synechococcus elongatus PCC 7942	CRISPR/dCas9	glgc、sdhA、sdhB	增加了琥珀酸产量	[53]
	Anabaena sp. PCC 7120	CRISPR/dCas9	glnA	成功微调glnA的表达水平;控制铵的生产。	[54]
	Synechocystis sp. PCC 6803	CRISPR/dCas9	PlsX	抑制必需的酰基转移酶PlsX可将脂肪醇滴度提高3倍。	[57]
	Synechococcus elongatus PCC 7942	CRISPR/dCpf1	nblA、acnB、cpcB2	使用CRISPR-dCas12a改善光合角鲨烯的生产	[56]
硅藻	Phaeodactylum tricornutum	CRISPR/Cas9	CpSRP54	CRISPR/Cas9首次在硅藻的应用	[58]
	Thalassiosira pseudonana	CRISPR/Cas9	urease	脲酶基因的精确删除	[59]
	Phaeodactylum tricornutum	CRISPR/Cas9	vtc2、pho4	优化方法,产生单等位基因突变和双等位基因突变	[60]
	Thalassiosira pseudonana	CRISPR/Cas9	TpθCA3	使用Cas9切口酶介导的基因组编辑,获得了海洋硅藻突变体	[61]
	Phaeodactylum tricornutum	CRISPR/Cas9	NR、GS-2、cGOGAT	新的Cas9附加体设计减少了生产和筛选突变菌株的时间	[62]
其他	Nannochloropsis oceanica IMET1	CRISPR/Cas9	Nitrate reductase gene、HygR	含油微藻的基因编辑,突变菌株敲除效率提高	[63]
	Nannochloropsis gaditana	CRISPR/Cas9	ZnCys	微调ZnCys表达,优化脂质生产	[64]
	Chlorella vulgarisFSP-E	CRISPR/Cas9	fad3	CRISPR / Cas9系统是首次应用于小球藻FSP-E	[65]